Antibodies and Elisas for Alpha Klotho

ABSTRACT

An antibody and/or binding fragment thereof, wherein the antibody and/or binding fragment thereof comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprising complementarity determining region (CDR) CDR-L3 and the heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3, with the amino acid sequences of said CDRs comprising one or more of the sequences set forth below: CDR-L3: selected from any one of SEQ ID NOs: 123, 126-130, 142, 148 or 149; CDR-H1: SEQ ID NOs: 121 or 124; CDR-H2; SEQ ID NOs: 122 or 125; and/or CDR-H3: selected from any one of SEQ ID NOs: 196-226.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 16/075,033,filed Aug. 2, 2018, which is a U.S. national phase application based onPCT Application No. PCT/CA2017/050127, filed Feb. 3, 2017, which claimsthe benefit of 35 U.S.C. § 119 based on the priority of U.S. ProvisionalPatent Application No. 62/290,776, filed Feb. 3, 2016, which isincorporated herein by reference in its entirety.

This invention was made with government support under grant numbersDK079328 and DK091392 awarded by The National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD

This disclosure relates to antibodies specific for folded forms ofαKlotho as well as methods and assays, for example, enzyme-linkedimmunosorbent assays, for detecting αKlotho.

BACKGROUND

The klotho gene was originally identified as a suppressor of prematureaging [1, reviewed in 2]. Klotho is a single-pass transmembrane proteinexpressed predominantly in the kidney, the parathyroid gland, and thechoroid plexus [1, 3, 4]. Paralogous proteins with distinct functionsand expression profiles, termed βKlotho and γKlotho [5, 6] are alsoknown.

αKlotho has diverse effects, including regulating ion transport, Wnt andinsulin signaling, renin-angiotensin system, recruitment of stem cells,anti-carcinogenesis, anti-fibrosis, and antioxidation. The highest levelof expression of αKlotho is in the kidney [1, 7, 8]. In addition to itstransmembrane form which is a co-receptor for fibroblast growth factor(FGF) 23 [9-11], αKlotho is also released into the circulation, urine,and cerebrospinal fluid as an endocrine substance [7, 12, 13] generatedby transcript splicing into a truncated peptide[2] or proteolyticrelease by secretases [14, 15]. A substantial portion of the circulatingαKlotho is nephrogenic in origin [16]. The phenotypic similaritiesbetween genetic αKlotho ablation and chronic kidney disease (CKD)support the notion that reduced renal expression of αKlotho ispathogenic [1, 16].

Reduced renal αKlotho transcript or protein levels [12,18-24] and serumαKlotho concentration [12, 20] was demonstrated in rodent CKD fromnephron reduction surgery, ischemia reperfusion injury, immune complexglomerulonephritis, polygenic or hormonal hypertension, metabolicsyndrome, and diabetes [12, 18-24]. This convergence suggests thatαKlotho deficiency may be a generic consequence of nephron loss. αKlothoreduction is potentially a sensitive and early biomarker of CKD and alsoprognostic of CKD complications [22]. Restoration of αKlotho inexperimental CKD in rodents ameliorates the kidney disease andextra-renal complications [12, 22, 23]. αKlotho deficiency has also beendocumented in acute kidney injury (AKI) in both rodents and humans [25].αKlotho can potentially serve as an early biomarker for AKI as it isreduced much earlier than changes in the current known biomarkers of AKI[26].

αKlotho forms a constitutive binary complex with FGF receptors (FGFRs)to confer selective affinity to FGF23 [10, 27]. Defects in αKlothoexpression result in FGF23 resistance and phosphate retention in mice[1, 28] and humans [29]. Therefore, αKlotho and FGF23 have emerged asessential components of the bone-kidney endocrine axis that regulatesphosphate metabolism [30, 31].

The extracellular domain of the membrane-anchored form of αKlotho can besecreted as a soluble protein. The soluble form is generated from themembrane-anchored form by membrane-anchored proteases and is releasedinto blood and urine [13, 15]. As noted above, membrane-anchored αKlothofunctions as part of the FGF23 receptor complex, whereas secretedαKlotho functions as an endocrine factor that exerts actions on distantorgans to exert highly pleiotropic actions as stated above (regulatingion transport, Wnt and insulin signaling, renin-angiotensin system,recruitment of stem cells, anti-carcinogenesis, anti-fibrosis, andantioxidation) [7].

Advanced CKD (Stages 4-5), characterized by kidney damage and decreasedkidney function, affects an estimated 2.6 million Canadians, greaterthan 7% of the population. A recent analysis of National VitalStatistics Report, National Health and Nutrition Examination Surveys andUS Renal Data System showed that the lifetime risks for white men, whitewomen, black men, and black women, are respectively: CKD stage 3a+,53.6%, 64.9%, 51.8%, and 63.6% [84]. The impact and burden of CKD andits associated complications on people's lives and the health caresystem is significant and will worsen in coming years [32-34]. Currentapproaches to treat CKD include modification of risk factors by diet andmedication, and for end stage renal disease (ESRD) by dialysis, andorgan replacement. There is an urgent need for additional therapies toarrest or delay progression of CKD at early stages, before complicationsarise. The majority of the complications of CKD are embraced within theentity of CKD-mineral bone disturbance (CKD-MBD) which are tied todisturbances of mineral metabolism. Phosphate retention is universallyobserved in CKD patients and associated with poor outcome [35, 36].Hyperphosphatemia is usually detected only in advanced stages of CKDwhen the disease is destined to progress to end-stage [37]. Recently, ithas been discovered that reduced renal αKlotho expression is one of theearliest events in CKD [12].

At present, there are some αKlotho antibodies and diagnostic kitsavailable on the market, but the existing αKlotho antibodies are not ofsufficient specificity and not efficient at immunoprecipitating αKlothofrom human serum, and the current immune-based assays for αKlotho arecostly and inadequate in sensitivity and specificity.

Low αKlotho transcript and protein levels have been described in humankidney from nephrectomy samples of end stage kidneys and biopsies frompatients with CKD [21,38]. Studies using an immune-based assay haveshown widely disparate results in terms of absolute values of serumαKlotho concentration (100-fold span in levels from different labs) anddirection of change (increased, decreased, or no change) with CKD andage [21,39-60]. The discrepant database has thwarted progress andincapacitated the ability to determine whether the promising rodent datacan be translated into meaningful human application. In addition to CKD,acute kidney injury (AKI) from a variety of causes is also associatedwith rapid decrease of αKlotho in the kidney [25, 61-65] and serum inrodents and in urine in humans [25]. There is no data on human serumαKlotho in AKI to date. There is a need for an early, sensitive, and/orspecific marker for renal injury in humans [66].

Generating antibodies to conserved proteins is challenging, as animalimmunization methods for antibody development are subject to mechanismsthat protect against auto-immunity. Synthetic antibody technology offersa powerful alternative because it is applied under defined in vitroconditions, uses antibody libraries that have not been subjected totolerance selection that remove self-reactive antibodies, and is provento yield antibodies with high affinities and specificities [67-71].Within an optimized antibody framework, sequence diversity is introducedinto the complementary determining regions (CDR's) by combinatorialmutagenesis. These libraries are coupled with phage display, with eachphage particle displaying a unique antigen-binding fragment (Fab) on itssurface while carrying the encoding DNA internally, thus achievingdirect phenotype-genotype relations. Fab-displaying phage that bind toan antigen of interest are enriched using binding selections withpurified antigens on solid support. The CDR's of binding phage clonesare identified by DNA sequencing and the Fab proteins are purified frombacteria, or converted to the full-length IgG in mammalian cells.

Barker et al. 2015 [86] describe an antibody having specific bindingaffinity with αKlotho, sb106, which was isolated following rounds ofbiopanning of a synthetic human Fab phase-displayed library. The sb106antibody has a binding affinity to αKlotho in the single-digit nanomolarrange and comprises the following CDR sequences (IMGT CDR residues areunderlined, IMGT framework region residues are not underlined andresidues at IMGT positions which were randomized in the selectionlibrary are shown in bold):

(CDR-L1) QSVSSA, (CDR-L2) SAS, (CDR-L3) QQAGYSPIT, (CDR-H1) GFNISYYS I,(CDR-H2) Y ISPSYGYT S and   (CDR-H3) ARYYVYASHGWAGYGMDY.

Additional αKlotho specific antibodies which bind a different epitopethan sb106 are desirable.

SUMMARY

This present disclosure relates to an antibody and/or binding fragmentthereof that comprises a light chain variable region and a heavy chainvariable region, the light chain variable region comprising acomplementarity determining region (CDR) CDR-L3 and the heavy chainvariable region comprising CDR-H1, CDR-H2 and CDR-H3, with the aminoacid sequences of said CDRs comprising one or more of the sequences setforth below:

-   -   CDR-L3: selected from any one of SEQ ID NOs: 123, 126-130, 142,        148 or 149;    -   CDR-H1: SEQ ID NOs: 121 or 124;    -   CDR-H2: SEQ ID NOs: 122 or 125; and/or    -   CDR-H3: selected from any one of SEQ ID NOs: 196-226.

In another embodiment, the antibody and/or binding fragment thereofcomprises CDRs having amino acid sequences selected from SEQ ID NOs:142-226, optionally as set forth below:

Light chain variable region:

-   -   CDR-L3: selected from any one of SEQ ID NOs: 142-156;

Heavy chain variable region:

-   -   CDR-H1: selected from any one of SEQ ID NOs: 157-174;    -   CDR-H2: selected from any one of SEQ ID NOs: 175-195; and/or    -   CDR-H3: selected from any one of SEQ ID NOs: 196-226.

In a further embodiment, the antibody and/or binding fragment thereofcomprises a light chain variable region comprising CDR-L1 and/or CDR-L2having the amino acid sequences of SEQ ID NO: 140 and SEQ ID NO: 141,respectively.

In an embodiment, the αKlotho polypeptide specifically bound by theantibody is a folded αKlotho polypeptide.

Another aspect includes a nucleic acid encoding an antibody and/orbinding fragment thereof described herein.

A further aspect is a vector comprising a nucleic acid described herein.

Another aspect includes a recombinant cell producing an antibody and/orbinding fragment thereof, nucleic acid or vector described herein.

Another aspect is an immunoassay comprising or using one or moreantibodies and/or binding fragments thereof described herein.

In an embodiment, the immunoassay is an enzyme linked immunosorbentassay (ELISA).

Other aspects include a method for producing an antibody and/or bindingfragment thereof, an assay for measuring the level of αKlothopolypeptide in a sample, an assay for detecting and/or measuring solubleαKlotho polypeptide as well as methods for screening, for diagnosing orfor detecting a kidney condition selected from chronic kidney disease(CKD) and acute kidney injury (AKI) in a subject, and methods ofprognosing disease progression and/or recovery.

Other features and advantages of the present disclosure will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the disclosure aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

An embodiment of the present disclosure will now be described inrelation to the drawings in which:

FIG. 1 shows the sequence, specificity and affinity of sb106. (A) CDRsequences and VH domain framework region residues variable in Library Ffor anti-αKlotho sb106, according to the IMGT (internationalImMunoGeneTics database) numbering scheme. IMGT CDR residues areunderlined, IMGT framework region residues are not underlined, residuesat IMGT positions which were randomized in the selection library arebold. (B) Specificity determination of anti-αKlotho sb106 by Fab-phageELISA: sb106 Fab-phage were incubated with the following immobilizedantigens: a complex of FGFR1c/αKlotho complex (aKL-R1), FGFR1c alone(R1), human αKlotho (Hu aKL) and mouse αKlotho (Mu aKL), or neutravidin(NA) and bovine serum albumin (BSA) as negative controls. After washingoff unbound phage, bound phages were detected using an HRP-conjugatedanti-phage antibody. Colorimetric HRP reagents allow for absorbancereadings at 450 nm. (C) Estimation of affinity by competitive Fab-phageELISA. sb106 Fab-phage were pre-incubated with 50, 5, 0.5, 0.05, 0.005and 0.0005 nM soluble human αKlotho. The binding signals to immobilizedhuman αKlotho reported are an average of two data sets. The reduction inbinding to immobilized αKlotho is indicative of the fraction bound tosoluble αKlotho, thus a 50% reduction in signal occurs when the solubleαKlotho concentration is approximately equal to the K_(D) of theinteraction.

FIG. 2 shows the characterization of sb106-Fab by immunoblot,immunohistochemistry and immunocytochemistry. (A) Immunoblot of kidneylysate from wild type mice (WT), homozygous αKlotho hypomorphic mice(kl/kl) and transgenic αKlotho overexpressing mice (Tg-KI), using themonoclonal antibody KM2076 or the sb106-Fab. GAPDH: Glyceraldehydephosphate dehydrogenase. (B) Immunoblot of lysates from normal ratkidney (NRK) cells, human embryonic kidney (HEK) cells, and HEK cellstransfected with a plasmid for over-expression of αKlotho, using themonoclonal antibody KM2076 or the sb106-Fab. (C) Fresh or fixed ratparathyroid tissue probed with phalloidin for β-actin or sb106-IgG. (D)sb106 immunostaining of HEK293T cells transfected with a vector control,or vector for over-expression for αKlotho or βKlotho. Representativecells are shown. The DAPI nuclear staining is labeled “N”. (scale bar,10 μm). αKlotho staining with Fab sb106 was only observed in cellstransfected with αKlotho and not in cell transfected with βKlotho.

FIG. 3 shows the characterization of sb106-Fab by immunoprecipitation.(A) HEK293 cells were transfected with empty vector or varyingquantities (μg/dish) of vector for expression of transmembrane fulllength αKlotho (TM-αKlotho) or soluble extracellular domain of αKlothowith a C-terminal FLAG epitope (s-αKlotho-FLAG). Cell lysates or cellculture medium was immunoprecipitated (IP) with either sb106-Fab oranti-FLAG MAb. Immunocomplexes were resolved by SDS-PAGE andimmunoblotted (IB) with monoclonal anti-αKlotho antibody KM2076. (B)Urine from rat, mouse, or human was immunoprecipitated with sb106-Fab,resolved by SDS-PAGE and immunoblotted (IB) with KM2076 (left threelanes). Size-selected urine (100 kDa cut-off) was directly subjected toSDS-PAGE and immunoblotted (right three lanes). (C) Immunoprecipitationsof endogenous αKlotho from serum. Serum samples from wild type (WT)mouse, klotho^(−/−) mouse, normal human, and dialysis patient (ESRD)where incubated with sb106-Fab overnight at 4° C. Sepharose beadsconjugated with anti-FLAG antibody were then added and incubated for 2hours at 4° C. The beads were washed and bound proteins were eluted with2×SDS sample loading buffer. Immunoblot was performed KM2076 followed bya standard anti-rat IgG secondary for visualization.

FIG. 4 shows the validation of IP-IB assay using human serum spiked withrecombinant αKlotho. (A) Known amounts of soluble human αKlothoectodomain were added to sera from a healthy volunteer or an anephricdialysis patient (CKD patient). αKlotho was measured in the sera usingthe IP-IB assay. (B) Similar experiment as in (A) except comparisonswere made where protease inhibitors (AEBSF 0.1 mM, aprotinin 0.3 μM,bestatin 10 μM, E-64 1 μM, leupeptin 50 μM, pepstatin A 1 μM) wereeither included or excluded from the IP. (C) αKlotho levels determinedby IP-IB (y-axis) were plotted against the added recombinant αKlotho(x-axis) in the four conditions described above. Extrapolation to zerospiking shows the level of endogenous αKlotho in the serum treated withprotease inhibitors. Only one line is shown for healthy serum with orwithout protease inhibitors as the results were indistinguishable.

FIG. 5 shows IP-IB assay of serum αKlotho in humans with chronic kidneydisease. (A) αKlotho was measured by the IP-IB assay in human sera fromnormal healthy volunteers and patients from a CKD clinic and dialysisunit using the conventional numerical staging using recombinant αKlothoas a calibration curve. Bars and error bars denote means and standarddeviations. The data was analyzed by ANOVA followed byStudent-Newman-Keuls test for pairwise multiple comparisons. P valuesachieving statistical significance between groups are indicated abovethe brackets. The number of subjects in each group is indicated at thebottom. (B) The concentrations of αKlotho in a large variety of humansera were determined either by IP-IB (x-axis) or by a commercial ELISA(y-axis) in the same samples. The dotted line represents identity. Thegrey diamonds represent sera that have been through one or morefreeze-thaw cycles (stored) and the black diamonds represent sera thawedonly once (fresh). (C) Sera from human subjects were assayed by IP-IBand ELISA. The same sera were subjected to the indicated cycles ofrepeated freeze-thaw and then assayed. Results for each sample wereexpressed as a percentage of the reading from the same sample thawedonly once. The black lines denote the mean of the different subjects.

FIG. 6 shows human urinary αKlotho levels. αKlotho was measured in theurine of healthy volunteers or patients with chronic kidney diseasestage 5 (CKD5). (A) A representative IP-IB assay using recombinantmurine αKlotho (rMKI) as a calibration with four subjects in each groupunder steady state conditions. Equal amounts of urine creatinine wereused for IP-IB. (B) Summary of the data from the IP-IB assay and thecommercial ELISA. Bars and error bars represent mean and standarddeviation from eight subjects in each group. The mean of the healthyvolunteers was set as a reference of 100%.

FIG. 7 shows surface plasmon resonance (SPR) sensorgrams with sb106-Fab.(A) SPR sensorgram illustrating binding of sb106-Fab (Fsb106) to theαKlotho-FGFR1c complex. The binary complex of murine αKlotho ectodomainand human FGFR1c ligand-binding domain was immobilized on a biosensorchip and 100 nM of Fsb106 were injected over the chip. Note that theFsb106 dissociates extremely slowly from the αKlotho-FGFR1c complex. (B)Overlay of SPR sensorgrams showing that sb106-Fab does not inhibitternary complex formation between FGF23, αKlotho, and FGFR1c. 10 nM ofαKlotho-FGFR1c complex alone and a mixture of 10 nM of αKlotho-FGFR1ccomplex and 100 nM of Fsb106 were injected over a biosensor chipcontaining immobilized FGF23.

FIG. 8 is a schematic of amino acid sequences of sb106. (A) Light chainsequence (SEQ ID NO: 11) of sb106. (B) Heavy chain sequence-Fab (SEQ IDNO: 12). (C) Heavy chain sequence—IgG1 (SEQ ID NO: 13). (D) Heavy chainsequence—IgG4 (SEQ ID NO: 14). IMGT CDR residues are underlined, IMGTframework region residues are not underlined, and residues at IMGTpositions which were randomized in the selection library are in bold andlarger font size, and italicized amino acids are constant domains.

FIG. 9 is graph showing absorbance values of a Fab phage ELISAdemonstrating the binding of Fab phage clones to αKlotho in the absenceor presence of sb106 Fab. Fab phage clones were incubated withimmobilized αKlotho in the absence or presence of sb106 Fab (25 ug/ml),or neutravidin (NA) as a negative control. After washing off unboundphage, bound phage were detected using an HRP-conjugated anti phageantibody. Colorimetric HRP reagents allow for absorbance readings at 450nm.

FIG. 10 is a graph showing absorbance values of Fab ELISAs withadditional αKlotho antibodies. Purified Fabs [5 ug/ml] were incubatedwith immobilized αKlotho (1 ug/ml) or bovine serum albumin (BSA), as anegative control. After washing off unbound Fab, bound Fab were detectedusing an HRP—conjugated anti-Flag anti-body (Fabs have Flag-tag on thelight chain). Colorimetric HRP reagents allow for absorbance readings at450 nm.

FIG. 11 is a series of graphs of epitope grouping experiments ofadditional αKlotho antibodies. αKlotho antigen is coated on to plates,blocked and then incubated with 10 ug/ml of Fab (as indicated on the xaxis) for 1 hour. Unbound Fab is washed and then the antigen with boundFab is exposed to Fab-phage (indicated at the top of each graph) for 20minutes. After washing off unbound phage, bound phage were detectedusing an HRP-conjugated anti-phage antibody. Colorimetric HRP reagentsallow for absorbance readings at 450 nm. Fab is the control, referencesignal for no interference and the BSA signal is the background control.

FIG. 12 is a series of graphs of affinity estimates measured by surfaceplasmon resonance with αKlotho Fabs. Fabs were immobilized using ananti-H+L IgG for capture. αKlotho injections were serially diluted 3times with a two-fold reduction each time; 50 ug/ml (highest curve) wasthe starting concentration and 6.25 ug/ml (lowest curve) was the finalconcentration. Curves are fitted to the Langmuir model.

FIG. 13 is a series of immunoblots showing immunoprecipitations ofαKlotho with identified Fabs.

FIG. 14 is a graph showing absorbance values of capture ELISAs with IgGsrepresenting 3 different epitopes of αKlotho.

FIG. 15 is series of graphs showing absorbance values of sandwich ELISAswith IgGs representing 3 different epitopes of αKlotho.

FIG. 16 is a graph showing absorbance values of a Fab ELISA using bothhuman and mouse αKlotho.

FIG. 17 is a series of graphs of affinity estimates measured by surfaceplasmon resonance against human and mouse antigen for select αKlothoFabs. Fabs were captured using an anti-IgG(H+L) antibody and serialdilutions of αKlotho were injected. Binding curves were fitted to theLangmuir model. FIG. 17(A) shows determined K_(on), K_(off) and K_(D)values. FIGS. 17 (B) and (C) show binding curves for human and mouseantigen respectively.

DETAILED DESCRIPTION OF THE DISCLOSURE I. Definitions

The term “αKlotho” or “alphaKlotho” as used herein refers to all knownand naturally occurring αKlotho molecules including, full length αKlothoprotein, fragments thereof such as ectodomain fragments, as well asnucleic acids encoding said protein and fragments, as determinable fromthe context used. Included are the soluble forms of αKlotho(proteolytically cleaved as well as alternatively spliced forms αKlotho,referred to as “soluble αKlotho” when present in a biological fluid suchas blood or a fraction thereof, urine or cerebrospinal fluid and havinga molecular weight of about 130 kDa, as well as the membrane-anchoredform of αKlotho, and including but not limited to mammalian αKlotho suchas human αKlotho, or rodent αKlotho including for example mouse and ratαKlotho.

The term “acute kidney injury” or “AKI” as used herein refers to anabrupt and sustained loss of kidney function for example that can leadto accumulation of urea and other chemicals in the blood, that developswithin for example seven days of an insult. AKI may be caused bydisease, injury such as crushing injury to skeletal muscle andmedication. AKI is classified in stages varying from risk (glomerularfiltration rate (GFR) decreased by 25%), injury (GFR decreased by 50%),failure (GFR decreased by 75%), loss (complete loss of kidney functionfor more than four weeks) and end-stage renal disease (complete loss ofkidney function for more than three months). AKI can be asymptomatic.

The term “early acute kidney injury” as used herein means priorto risesin serum creatinine.

The term “amino acid” includes all of the naturally occurring aminoacids as well as modified amino acids.

The term “antibody” as used herein is intended to include humanantibodies, monoclonal antibodies, polyclonal antibodies, single chainand other chimeric antibodies. The antibody may be from recombinantsources and/or produced in transgenic animals. The antibody in anembodiment comprises a heavy chain variable region or a heavy chaincomprising a heavy chain complementarity determining region 1, heavychain complementarity determining region 2 and heavy chaincomplementarity determining region 3, as well as a light chain variableregion or light chain comprising a light chain complementaritydetermining region 1, light chain complementarity determining region 2and light chain complementarity determining region 3.

The term “binding fragment” as used herein is intended to includewithout limitations Fab, Fab′, F(ab′)2, scFv, scFab, dsFv, ds-scFv,dimers (e.g. Fc dimers), minibodies, diabodies, and multimers thereof,multispecific antibody fragments and Domain Antibodies. Antibodies canbe fragmented using conventional techniques. For example, F(ab′)2fragments can be generated by treating the antibody with pepsin. Theresulting F(ab′)2 fragment can be treated to reduce disulfide bridges toproduce Fab′ fragments. Papain digestion can lead to the formation ofFab fragments. Fab, Fab′ and F(ab′)2, scFv, scFab, dsFv, ds-scFv,dimers, minibodies, diabodies, bispecific antibody fragments and otherfragments can also be synthesized by recombinant techniques.

The term “capture antibody” as used herein means an antibody or bindingfragment thereof bound to a solid support and used to capture the targetantigen in a sample, for example αKlotho polypeptide, optionally solubleαKlotho polypeptide by forming a complex with the target antigen.

The term “detection antibody” as used herein means an antibody orbinding fragment thereof that binds a target antigen, for exampleαKlotho polypeptide, optionally soluble αKlotho polypeptide, optionallya target antigen already in a complex with a capture antibody. Forexample, the detection antibody binds the capture antibody:αKlothocomplex at an epitope on the target antigen that is different than theone recognized by the capture antibody.

A “conservative amino acid substitution” as used herein, is one in whichone amino acid residue is replaced with another amino acid residuewithout abolishing the protein's desired properties. Suitableconservative amino acid substitutions can be made by substituting aminoacids with similar hydrophobicity, polarity, and R-chain length for oneanother. Examples of conservative amino acid substitution include:

Conservative Substitutions Type of Amino Acid Substitutable Amino AcidsHydrophilic Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, Thr Sulphydryl CysAliphatic Val, Ile, Leu, Met Basic Lys, Arg, His Aromatic Phe, Tyr, Trp

The term “chronic kidney disease” or “CKD” refers to a disease causing aprogressive loss in renal function. CDK is classified according to fivestages which are determined according to a defined glomerular filtrationrate (GFR). Stage 1 CKD is defined by a GFR of ≥90 mL/min/1.73 m², stage2 CDK is defined by a GFR between 60-89 mL/min/1.73 m², stage 3 CKD isdefined by a GFR between 30-59 mL/min/1.73 m², stage 4 CKD is defined bya GFR between 15-29 mL/min/1.73 m² and stage 5 CKD is defined by a GFRof less than 15 mL/min/1.73 m². Normal kidney function is defined by aGFR between 100-130 mL/min/1.73 m² or 90 mL/min/1.73 m² withoutproteinuria.

The term “control” as used herein refers to a sample from a subject or agroup of subjects who are either known as having a kidney disease or nothaving the disease, and/or a value determined from said group ofsubjects, wherein subjects with αKlotho level at or below such value arelikely to have the disease. The disease can be for example chronickidney disease (CKD) or acute kidney injury (AKI). The disease can alsobe for example a stage of CKD such as stage 1 CKD, stage 2 CKD, stage 3CKD, stage 4 CKD or stage 5 CKD; higher stage being more severe. Inaddition, the control can be for example derived from tissue of the sametype as the sample of the subject being tested. In methods directed tomonitoring, the control can also be tissue from the same subject takenat different a time point for example the control can be a sample fromthe same subject taken prior to a treatment for a kidney disease.

The term “early chronic kidney disease” refers to earlier stages of CKD,and means in an embodiment stage 1 and/or stage 2 CKD. Frequently, thereare no elevations of FGF23, PTH, and phosphate. Subjects with stage 1CKD almost never present any symptoms indicating kidney damage. Subjectswith stage 2 CKD do not necessarily present symptoms indicating kidneydamage but occasionally do.

The term “denatured” as used herein means a polypeptide that has losttertiary and/or secondary structure (e.g. fully unfolded protein), forexample when exposed to denaturing conditions in SDS sample loadingbuffer.

The term “detectable tag” as used herein refers to moieties such aspeptide sequences that can be appended or introduced into recombinantprotein.

The term “sandwich ELISA” as used herein refers to an ELISA comprising asolid support and a capture antibody or binding fragment thereof(specific for the antigen) immobilized onto the solid support. In suchan ELISA an amount of target antigen in a sample is bound by the captureantibody (e.g. αKlotho polypeptide comprised in a sample). The boundantigen is detected by a second antibody or binding fragment thereof,i.e. a detection antibody or binding fragment thereof, which recognizesan epitope that is different from the one recognized by the captureantibody. The capture antibody:αKlotho complex) is detected by thedetection antibody which can be covalently linked to an enzyme or canitself be detected by addition of a secondary antibody which is linkedto an enzyme. For example, the capture antibody and/or the detectionantibody can comprise CDR regions disclosed herein.

The term “epitope” as used herein refers to the site on the antigen thatis recognized by the antibodies or binding fragments disclosed herein.

The term “heavy chain complementarity determining region” as used hereinrefers to regions of hypervariability within the heavy chain variableregion of an antibody molecule. The heavy chain variable region hasthree complementarity determining regions termed heavy chaincomplementarity determining region 1 (CDR-H1), heavy chaincomplementarity determining region 2 (CDR-H2) and heavy chaincomplementarity determining region 3 (CDR-H3) from the amino terminus tocarboxy terminus. All CDRs and framework regions (FRs) disclosed herein,amino acid sequences of CDRs and FRs disclosed herein, and CDR-encodingor FR-encoding nucleic acid sequences disclosed herein, are intended tobe defined in accordance with IMGT numbering (85).

The term “heavy chain variable region” as used herein refers to thevariable domain of the heavy chain comprising the heavy chaincomplementarity determining region 1, heavy chain complementaritydetermining region 2 and heavy chain complementarity determining region3. One or more amino acids or nucleotides can be modified for examplereplaced with a conservative substitution, for example outside the CDRsequences.

The term “host cell” refers to a cell into which a recombinant DNAexpression vector can be introduced to produce a recombinant cell. Thehost cell can be a bacterial cell such as E. coli but can also be anytype of microbial, yeast, fungi, insect or mammalian host cell.

The term “isolated antibody or binding fragment thereof” or “isolatedand purified antibody or binding fragment thereof” refers to an antibodyor binding fragment thereof that is substantially free of cellularmaterial or culture medium when produced by recombinant DNA techniques,or chemical precursors, or other chemicals when chemically synthesizedand/or other antibodies, for example directed to a different epitope.

The term “K_(D)” refers to the dissociation constant of a complex forexample of a particular antibody-antigen interaction.

The term “light chain complementarity determining region” as used hereinrefers to regions of hypervariability within the light chain variableregion of an antibody molecule. Light chain variable regions have threecomplementarity determining regions termed light chain complementaritydetermining region 1, light chain complementarity determining region 2and light chain complementarity determining region 3 from the aminoterminus to the carboxy terminus.

The term “light chain variable region” as used herein refers to thevariable domain of the light chain comprising the light chaincomplementarity determining region 1, light chain complementaritydetermining region 2 and light chain complementarity determining region3.

The term “native” or “natively folded” as used herein refers to aprotein in its native conformation (e.g. 3D conformation) or in aconformation sufficient to confer functionality, including for examplepartially unfolded protein capable of binding a receptor or ligand. Forexample, folded αKlotho protein is capable of binding to a FGF receptorsuch as FGFR1c and can form a FGFR1c: αKlotho complex.

The term “nucleic acid sequence” as used herein refers to a sequence ofnucleoside or nucleotide monomers consisting of naturally occurringbases, sugars and intersugar (backbone) linkages. The term also includesmodified or substituted sequences comprising non-naturally occurringmonomers or portions thereof. The nucleic acid sequences of the presentapplication may be deoxyribonucleic acid sequences (DNA) or ribonucleicacid sequences (RNA) and may include naturally occurring bases includingadenine, guanine, cytosine, thymidine and uracil. The sequences may alsocontain modified bases. Examples of such modified bases include aza anddeaza adenine, guanine, cytosine, thymidine and uracil; and xanthine andhypoxanthine. The nucleic acid can be either double stranded or singlestranded, and represents the sense or antisense strand. Further, theterm “nucleic acid” includes the complementary nucleic acid sequences aswell as codon optimized or synonymous codon equivalents. The term“isolated nucleic acid sequences” as used herein refers to a nucleicacid substantially free of cellular material or culture medium whenproduced by recombinant DNA techniques, or chemical precursors, or otherchemicals when chemically synthesized. An isolated nucleic acid is alsosubstantially free of sequences which naturally flank the nucleic acid(i.e. sequences located at the 5′ and 3′ ends of the nucleic acid) fromwhich the nucleic acid is derived.

The term “polypeptide” as used herein refers to a polymer consisting ofa large number of amino acid residues bonded together in a chain. Thepolypeptide can form a part or the whole of a protein. The polypeptidemay be arranged in a long, continuous and unbranched peptide chain. Thepolypeptide may also be arranged in a biologically functional way. Thepolypeptide may be folded into a specific three dimensional structurethat confers it a defined activity. The term “polypeptide” as usedherein is used interchangeably with the term “protein”.

The term “isolated polypeptide” as used herein means substantially freeof cellular material or culture medium when produced by recombinant DNAtechniques, or chemical precursors, or other chemicals when chemicallysynthesized.

The term “reference agent” as used herein refers to an agent that can beused in an assay and that can be for example a standard amount ofαKlotho protein used as a reference for example for detecting, screeningor for diagnosing kidney condition such as chronic kidney disease andacute kidney disease.

The term “sample” as used herein refers to any biological fluid, cell ortissue sample from a subject, which can be assayed for αKlotho such assoluble biomarkers. For example the sample can comprise urine, serum,plasma or cerebrospinal fluid. The sample can for example be a“post-treatment” sample wherein the sample is obtained after one or moretreatments, or a “base-line sample” which is for example used as a baseline for assessing disease progression.

The term “sb106”, “sb106 antibody” or “clone ID 48” as used herein meansan antibody comprising light and heavy chain amino acid sequences setforth below:

Light chain variable region sequence (IMGT CDR sequences are underlined,IMGT framework region residues are not underlined, and residues at IMGTpositions which were randomized in the selection library are shown inbold):

(SEQ ID NO: 11) DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQAGYSPITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECHeavy chain variable region sequence (IMGT CDR sequences are underlined,IMGT framework region residues are not underlined, and residues at IMGTpositions which were randomized in the selection library are shown inbold):

(SEQ ID NO: 12) EVQLVESGGGLVQPGGSLRLSCAASGFNISYY SIHWVRQAPGKGLEWVA YISPSYGYT SYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYYVYASHGWAGYGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTand comprising complementarity determining regions determined using IMGTnumbering, e.g. with the amino acid sequences set forth below (IMGT CDRsequences are underlined, IMGT framework region residues are notunderlined, and residues at IMGT positions which were randomized in theselection library are shown in bold):

Light chain variable region:

CDR-L1: (SEQ ID NO: 9) QSVSSA CDR-L2: (SEQ ID NO: 10) SAS CDR-L3:(SEQ ID NO: 5) QQAGYSPIT

Heavy chain variable region:

CDR-H1: (SEQ ID NO: 6) GFNISYYSI CDR-H2: (SEQ ID NO: 7) Y ISPSYGYT SCDR-H3: (SEQ ID NO: 8) ARYYVYASHGWAGYGMDY.Sub-clones (e.g. variants) of sb106 were identified which have one ormore CDRs (as shown in Table 2) which replace corresponding CDRs ofsb106, and which have non-Library F-variable framework regions (i.e. allIMGT framework region positions except for IMGT VH domain positions 39,55 and 66) which are identical to those of sb106.

Sb106 recognizes an epitope of αKlotho that is different from an epitoperecognized by the antibodies and/or binding fragments herein disclosed(e.g. epitope B and epitope C). As such, sb106 and antibodies comprisingthe CDRs of sb106 and/or the variants of sb106 may be used inconjunction with the antibodies and/or binding fragments thereof hereindescribed (e.g. directed to epitope B and/or epitope C) in a detectionassay, for example an ELISA such as a sandwich ELISA.

The term “sequence identity” as used herein refers to the percentage ofsequence identity between two polypeptide sequences or two nucleic acidsequences. To determine the percent identity of two amino acid sequencesor of two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of afirst amino acid or nucleic acid sequence for optimal alignment with asecond amino acid or nucleic acid sequence). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=number of identical overlappingpositions/total number of positions.times.100%). In one embodiment, thetwo sequences are the same length. The determination of percent identitybetween two sequences can also be accomplished using a mathematicalalgorithm. A preferred, non-limiting example of a mathematical algorithmutilized for the comparison of two sequences is the algorithm of Karlinand Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modifiedas in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A.90:5873-5877. Such an algorithm is incorporated into the NBLAST andXBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLASTnucleotide searches can be performed with the NBLAST nucleotide programparameters set, e.g., for score=100, wordlength=12 to obtain nucleotidesequences homologous to a nucleic acid molecules of the presentapplication. BLAST protein searches can be performed with the XBLASTprogram parameters set, e.g., to score-50, wordlength=3 to obtain aminoacid sequences homologous to a protein molecule of the presentinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., 1997, NucleicAcids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to performan iterated search which detects distant relationships between molecules(Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, thedefault parameters of the respective programs (e.g., of XBLAST andNBLAST) can be used (see, e.g., the NCBI website). Another preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers and Miller, 1988,CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used. The percent identity between twosequences can be determined using techniques similar to those describedabove, with or without allowing gaps. In calculating percent identity,typically only exact matches are counted.

By “at least moderately stringent hybridization conditions” it is meantthat conditions are selected which promote selective hybridizationbetween two complementary nucleic acid molecules in solution.Hybridization may occur to all or a portion of a nucleic acid sequencemolecule. The hybridizing portion is typically at least 15 (e.g. 20, 25,30, 40 or 50) nucleotides in length. Those skilled in the art willrecognize that the stability of a nucleic acid duplex, or hybrids, isdetermined by the Tm, which in sodium containing buffers is a functionof the sodium ion concentration and temperature (Tm=81.5° C.−16.6 (Log10 [Na+])+0.41(%(G+C)−600/l), or similar equation). Accordingly, theparameters in the wash conditions that determine hybrid stability aresodium ion concentration and temperature. In order to identify moleculesthat are similar, but not identical, to a known nucleic acid molecule a1% mismatch may be assumed to result in about a 1° C. decrease in Tm,for example if nucleic acid molecules are sought that have a >95%identity, the final wash temperature will be reduced by about 5° C.Based on these considerations those skilled in the art will be able toreadily select appropriate hybridization conditions. In preferredembodiments, stringent hybridization conditions are selected. By way ofexample the following conditions may be employed to achieve stringenthybridization: hybridization at 5× sodium chloride/sodium citrate(SSC)/5×Denhardt's solution/1.0% SDS at Tm−5° C. based on the aboveequation, followed by a wash of 0.2×SSC/0.1% SDS at 60° C. Moderatelystringent hybridization conditions include a washing step in 3×SSC at42° C. It is understood, however, that equivalent stringencies may beachieved using alternative buffers, salts and temperatures. Additionalguidance regarding hybridization conditions may be found in: CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in:Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold SpringHarbor Laboratory Press, 2001.

The term “subject” as used herein refers to any member of the animalkingdom, preferably a mammal, more preferably a human being or a rodentsuch as a rat or a mouse. In one embodiment, the subject is suspected ofhaving a kidney disorder such as chronic kidney disease (CKD) or acutekidney injury (AKI).

The term “variant” as used herein includes one or more amino acid and/ornucleotide modifications in a sequence (polypeptide or nucleic acidrespectively) for example, one or more modifications of a light chain ora heavy chain complementarity determining region (CDR) disclosed hereinthat perform substantially the same function as the light chain andheavy chain CDRs disclosed herein in substantially the same way. Forinstance, variants of the CDRs disclosed herein have the same functionof being able to specifically bind to an epitope on folded αKlothoprotein or in the case of nucleotide modifications, encode CDRs thathave same function of being able to specifically bind to an epitope onfolded αKlotho protein. For example, codon optimized and degeneratesequences are included. Variants of CDRs disclosed herein include,without limitation, conservative amino acid substitutions as well asadditions and deletions to the CDR sequences disclosed herein. Forexample the addition or deletion can be 1, 2, 3 or 4 amino acids and/orthe corresponding number of nucleotides.

The term “level” as used herein refers to an amount (e.g. relativeamount or concentration) of αKlotho protein that is detectable ormeasurable in a sample. For example, the soluble αKlotho level can be aconcentration such as pM or a relative amount such as 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6,3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0 and/or 10 times a control level, wherefor example, the control level is the level of soluble αKlotho in ahealthy subject.

In understanding the scope of the present disclosure, the term“comprising” and its derivatives, as used herein, are intended to beopen ended terms that specify the presence of the stated features,elements, components, groups, integers, and/or steps, but do not excludethe presence of other unstated features, elements, components, groups,integers and/or steps. The foregoing also applies to words havingsimilar meanings such as the terms, “including”, “having” and theirderivatives. Finally, terms of degree such as “substantially”, “about”and “approximately” as used herein mean a reasonable amount of deviationof the modified term such that the end result is not significantlychanged. These terms of degree should be construed as including adeviation of at least ±5% of the modified term if this deviation wouldnot negate the meaning of the word it modifies.

In understanding the scope of the present disclosure, the term“consisting” and its derivatives, as used herein, are intended to beclose ended terms that specify the presence of stated features,elements, components, groups, integers, and/or steps, and also excludethe presence of other unstated features, elements, components, groups,integers and/or steps.

The recitation of numerical ranges by endpoints herein includes allnumbers and fractions subsumed within that range (e.g. 1 to 5 includes1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood thatall numbers and fractions thereof are presumed to be modified by theterm “about.” Further, it is to be understood that “a,” “an,” and “the”include plural referents unless the content clearly dictates otherwise.The term “about” means plus or minus 0.1 to 10%, 1-10%, or preferably1-5%, of the number to which reference is being made.

Further, the definitions and embodiments described in particularsections are intended to be applicable to other embodiments hereindescribed for which they are suitable as would be understood by a personskilled in the art. For example, in the following passages, differentaspects of the invention are defined in more detail. Each aspect sodefined may be combined with any other aspect or aspects unless clearlyindicated to the contrary. In particular, any feature indicated as beingpreferred or advantageous may be combined with any other feature orfeatures indicated as being preferred or advantageous.

II. Antibody and/or Binding Fragment Thereof

The present disclosure relates to an antibody and/or binding fragmentthereof and methods of making and use for example for diagnosing and/orprognosing kidney diseases.

International application No. PCT/CA2015/050728 entitled ANTIBODIES WITHHIGH AFFINITY FOR ALPHA-KLOTHO, herein incorporated by reference,disclosed sb106 antibody. Herein described are additional antibodiesthat specifically bind αKlotho at different epitopes. As described inFIG. 10, the additional antibodies were identified from a syntheticantibody library and were shown to bind αKlotho. As shown in Example 11and FIG. 11, epitope grouping experiments using competitive ELISA revealthat the sb106 antibody binds a distinct epitope (identified as A),while the presently described antibodies bind at least two differentepitopes, identified as epitopes B and C. It is further described inFIG. 14 that the epitopes A and B are located within amino acids 550-981of αKlotho whereas epitope C is located within amino acids 1-549 ofαKlotho. These experiments show that the 3 epitopes to which theantibodies bind can be used in a detection assay, for example an ELISA.

Accordingly, a first aspect is an antibody and/or binding fragmentthereof that specifically binds αKlotho polypeptide at a differentepitope than recognized by an antibody having light and heavy chainvariable regions comprising the amino acid sequences of SEQ ID NO: 11and 12, respectively.

In an embodiment, “CDR-H1” is composed of IMGT CDR-H1 and the VH domainresidue at IMGT position 39 flanking the carboxy terminal residue ofIMGT CDR-H1; and “CDR-H2” is composed of IMGT CDR-H2 and the VH domainresidues at IMGT positions 55 and 66 flanking the amino terminal residueand flanking the carboxy terminal residue, respectively, of IMGT CDR-H2.

In an embodiment, the antibody or binding fragment thereof has a lightchain variable region comprising CDR-L1, CDR-L2 and CDR-L3 and a heavychain variable region comprising CDR-H1, CDR-H2 and CDR-H3 wherein theCDR regions are determined using IMGT numbering.

In an embodiment, the antibody and/or binding fragment thereof bindsαKlotho polypeptide at a different epitope than recognized by anantibody or binding fragment thereof having a light chain variableregion comprising CDR-L3 having amino acid sequences SEQ ID NO: 5, and aheavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 havingamino acid sequences SEQ ID NO: 6, 7 and 8, respectively.

In an embodiment, the antibody and/or binding fragment thereof bindsαKlotho polypeptide at a different epitope than recognized by anantibody or binding fragment thereof having a light chain variableregion comprising CDR-L1, CDR-L2 and CDR-L3 having amino acid sequencesSEQ ID NO: 9, 10 and 5, respectively, and a heavy chain variable regioncomprising CDR-H1, CDR-H2 and CDR-H3 having amino acid sequences SEQ IDNO: 6, 7 and 8, respectively.

In one embodiment, the antibody and/or binding fragment thereof binds toepitope B located within amino acids 550-981 of αKlotho however does notbind to the epitope recognized by sb106 antibody.

In an embodiment, the antibody and/or binding fragment thereofspecifically binds within amino acids 1 to 549 of αKlotho polypeptide.

In an embodiment, the αKlotho polypeptide is folded, optionally innative conformation (e.g. fully folded).

Accordingly another aspect is an antibody and/or binding fragmentthereof, wherein the antibody and/or binding fragment thereofspecifically binds to an epitope of a folded αKlotho polypeptide andspecifically binds αKlotho polypeptide at a different epitope thanrecognized by an antibody having light and heavy chain variable regionscomprising the amino acid sequences of SEQ ID NO: 11 and 12,respectively.

A further aspect is an antibody and/or binding fragment thereof, whereinthe antibody and/or binding fragment thereof specifically binds toαKlotho polypeptide in an unfixed or mildly fixed sample andspecifically binds αKlotho polypeptide at a different epitope thanrecognized by an antibody having light and heavy chain variable regionscomprising the amino acid sequences of SEQ ID NO: 11 and 12,respectively.

In an embodiment, the αKlotho polypeptide in the unfixed or mildly fixedsample is folded αKlotho.

As shown in Example 12, the antibodies herein disclosed have bindingaffinities to αKlotho ranging from 240 μM to 8.7 nM. In an embodiment,the antibody and/or binding fragment has a dissociation constant (K_(D))for the αKlotho polypeptide of about or less than 50 nM, about or lessthan 40 nM, about or less than 30 nM, about or less than 25 nM, about orless than 20 nM, about or less than 15 nM, about or less than 12 nM,about or less than 10 nM, about or less than 9 nM, about or less than 8nM, about or less than 7 nM, about or less than 6 nM, about or less than5 nM, about or less than 4 nM, about or less than 3 nM, about or lessthan 2 nM or about or less than 1 nM, as measured by competitive ELISAassay and/or SPR immunoassay.

In an embodiment, the antibody and/or binding fragment thereof comprisesa light chain variable region and a heavy chain variable region, thelight chain variable region comprising complementarity determiningregion CDR-L3 and the heavy chain variable region comprisingcomplementarity determining regions CDR-H1, CDR-H2 and CDR-H3, with theamino acid sequences of said CDRs comprising one or more of thesequences set forth below:

CDR-L3: selected from any one of SEQ ID NOs: 123, 126-130, 142, 148 or149;

CDR-H1: SEQ ID NOs: 121 or 124;

CDR-H2: SEQ ID NOs: 122 or 125; and/or

CDR-H3: selected from any one of SEQ ID NO: 196-226.

In an embodiment, the CDR-H1 region comprises the sequence of SEQ ID NO:133 or 134.

In an embodiment, the CDR-H2 region comprises the sequence of SEQ ID NO:135 or 136.

In an embodiment, the complementarity determining regions comprise theamino acid sequences selected from SEQ ID NOs: 142-226, optionally asset forth below:

CDR-L3: selected from any one of SEQ ID NOs: 142-156;

CDR-H1: selected from any one of SEQ ID NOs: 157-174;

CDR-H2: selected from any one of SEQ ID NOs: 175-195; and/or

CDR-H3: selected from any one of SEQ ID NOs: 196-226.

In an embodiment, the light chain variable region further comprisescomplementarity determining regions CDR-L1 and/or CDR-L2 comprising theamino acid sequences set forth below:

CDR-L1: SEQ ID NO: 140 and/or

CDR-L2: SEQ ID NO: 141.

In an embodiment, the antibody and/or binding fragment thereof comprisesa light chain variable region and a heavy chain variable regioncomprising CDR-L1, -L2, -L3, -H1, -H2 and -H3 amino acid sequences asset forth in Tables 3A and 3D-31.

In an embodiment, the antibody and/or binding fragment thereof bindsepitope B of αKlotho and comprises CDR regions as set forth in Tables 3Aand/or 3D-3F.

In an embodiment, the antibody and/or binding fragment thereof thatspecifically binds αKlotho comprises CDR regions of an antibodyidentified as specific for epitope B as set forth in Table 3A.

In an embodiment, the CDRs of the antibody and/or binding fragmentthereof that specifically binds αKlotho are selected from thoseindicated for clones 4804, 4805, 4807, 4808, 4809, 4811, 4812, 4813,4815, 4816, 4818, 4820, 4821, 4822, 4823, 4824, 4825, 4826, 4827, 4829,4832, 4833 and 4834, as set forth in Table 3A.

In an embodiment, the antibody and/or binding fragment thereof thatspecifically binds αKlotho comprises CDR regions of an antibodyidentified as specific for epitope C as set forth in Table 3A and/or3G-3I.

In an embodiment, the antibody and/or binding fragment thereof thatspecifically binds αKlotho comprises CDR regions of an antibodyidentified as specific for epitope C, as set forth in Table 3A.

In an embodiment, the CDRs of the antibody and/or binding fragmentthereof that specifically binds αKlotho are selected from thoseindicated for clones 4814, 4819, 4830 and 4831, as set forth in Table3A.

In an embodiment, CDRs of the antibody and/or binding fragment thatspecifically binds αKlotho are selected from those indicated for clones4806, 4810, 4817 and 4828, as set forth in Table 3A.

As an example, the CDR sequences of antibody sb173 (clone id 4808) inthe context of the full length light and heavy variable regions areshown in Table 4. The underlined residues denoting the CDR regions maybe replaced with other CDR sequences described herein, for example asset forth in Table 3A.

The antibody optionally a human antibody can be any class ofimmunoglobulins including: IgM, IgG, IgD, IgA or IgE; and any isotype,including: IgG1, IgG2, IgG3 and IgG4.

Any of the Fab clones can for example be inserted into a full lengthimmunoglobulin molecule, for example by subcloning. CDRs of a Fab cloneidentified here can be grafted onto an antibody to make a CDR-graftedantibody.

Humanized or chimeric antibody may include sequences from one or morethan one isotype or class.

Further, antibodies described herein may be produced as antigen bindingfragments such as Fab, Fab′ F(ab′)₂, Fd, Fv and single domain antibodyfragments, or as single chain antibodies in which the heavy and lightchains are linked by a spacer. Also, the human or chimeric antibodiesmay exist in monomeric or polymeric form.

Chimeric antibodies can be prepared using recombinant techniques. Asdescribed in the Examples, the Fab identified in the screen wasreformatted into full length IgG by subcloning the variable domains ofthe antibody's light and heavy chains into mammalian expression vectorsand producing the IgG protein for example as shown in the Examples usinghuman embryonic kidney cells (HEK293T). As described elsewhere any celltype suitable for expressing an antibody can be used.

In yet another embodiment, the light chain complementarity determiningregion CDR-L3 and heavy chain complementarity determining regionsCDR-H1, CDR-H2 and CDR-H3 have at least 70%, at least 80% or at least90% sequence identity to SEQ ID NOs: 142-156, SEQ ID NOs: 157-174, SEQID NOs: 175-195, and SEQ ID NOs: 196-226, respectively.

In an embodiment, the antibody, binding fragment thereof, optionally theCDR sequence has one or more conservative substitutions.

In one embodiment, the antibody and/or binding fragment thereof isselected from the group consisting of a an immunoglobulin molecule, aFab, a Fab′, a F(ab)2, a F(ab′)2, a Fv, a disulfide linked Fv, a scFv, adisulfide linked scFv, a single chain domain antibody including fragmentscFab, a diabody, a dimer, a minibody, a bispecific antibody fragment, achimeric antibody, a human antibody, a humanized antibody and apolyclonal antibody.

Fab, Fab′ and F(ab′)₂, scFv, scFab, dsFv, ds-scFv, dimers, minibodies,diabodies, bispecific antibody fragments and other fragments can besynthesized or expressed by recombinant techniques.

Antibodies can also be fragmented using conventional techniques. Forexample, F(ab′)₂ fragments can be generated by treating the antibodywith pepsin. The resulting F(ab′)₂ fragment can be treated to reducedisulfide bridges to produce Fab′ fragments. Papain digestion can leadto the formation of Fab fragments.

In an embodiment, the antibody is a human antibody.

Human antibodies are optionally obtained from transgenic animals (U.S.Pat. Nos. 6,150,584; 6,114,598; and 5,770,429). In this approach theheavy chain joining region (J_(H)) gene in a chimeric or germ-linemutant mouse is deleted. Human germ-line immunoglobulin gene array issubsequently transferred to such mutant mice. The resulting transgenicmouse is then capable of generating a full repertoire of humanantibodies upon antigen challenge.

In an embodiment, the antibody is a chimeric antibody comprising one ormore CDRs selected from SEQ ID NOs: 140 to 226.

As shown in Example 15, the antibody and/or binding fragment thereofherein disclosed is cross-reactive to several species. In an embodiment,the αKlotho polypeptide bound is mammalian αKlotho polypeptide, forexample, the αKlotho polypeptide is selected from human αKlothopolypeptide or rodent αKlotho polypeptide such as mouse αKlothopolypeptide or rat αKlotho polypeptide.

In an embodiment, the antibody and/or binding fragment thereofpreferentially binds human αKlotho over mouse αKlotho.

In another embodiment, the folded αKlotho polypeptide is soluble foldedαKlotho polypeptide. For example, the antibody and/or binding fragmentthereof binds soluble folded αKlotho polypeptide found in urine, plasma,and/or serum.

As shown in Example 16, Fab fragments herein disclosed were able to bindαKlotho alone and in a complex (αKlotho-FGFR1c). In yet anotherembodiment, the antibody and/or binding fragment thereof binds a complexcomprising folded αKlotho polypeptide. For example, the complex cancomprise the folded αKlotho polypeptide with a fibroblast growth factor(FGF) receptor, optionally FGFR1c.

In a further embodiment, the antibody and/or binding fragment islabelled and/or conjugated to a tag, for example to produce a diagnosticagent. For example, the detectable tag can be a purification tag such asa His-tag, a HA-tag, a GST-tag, biotin or a FLAG-tag.

The label is preferably capable of producing, either directly orindirectly, a detectable signal. For example, the label may beradio-opaque or a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, ¹²³I, ¹²⁵I,¹³¹I; a fluorescent (fluorophore) or chemiluminescent (chromophore)compound, such as fluorescein isothiocyanate, rhodamine or luciferin; anenzyme, such as alkaline phosphatase, beta-galactosidase or horseradishperoxidase (HRP); an imaging agent; or a metal ion.

Another aspect of the disclosure relates to an antibody complexcomprising the antibody and/or binding fragment thereof and αKlotho,optionally further comprising FGFR1c.

In an embodiment, the antibody complex comprises FGFR1c and optionallyfurther comprises FGF23.

In an embodiment, the antibody and/or binding fragment thereof is anisolated antibody and/or binding fragment thereof.

Yet another aspect is a nucleic acid encoding an antibody and/or bindingfragment thereof such as a binding fragment thereof described herein. Inan embodiment, the nucleic acid encodes an antibody and/or bindingfragment thereof comprising a light chain variable region and a heavychain variable region, the light chain variable region comprisingcomplementarity determining regions CDR-L1, CDR-L2 and CDR-L3 and theheavy chain variable region comprising complementarity determiningregions CDR-H1, CDR-H2 and CDR-H3, with the amino acid sequences of saidCDRs comprising one or more of the sequences set forth below:

CDR-L3: selected from any one of SEQ ID NOs: 123, 126-130, 142, 148 or149;

CDR-H1: SEQ ID NOs: 121 or 124;

CDR-H2: SEQ ID NOs: 122 or 125; and/or

CDR-H3: selected from any one of SEQ ID NOs: 196-226.

In an embodiment, the nucleic acid encoding an antibody and/or bindingfragment thereof further comprises the sequences set forth below:

CDR-L3: selected from any one of SEQ ID NOs: 229-243;

CDR-H1: selected from any one of SEQ ID NOs: 244-262;

CDR-H2: selected from any one of SEQ ID NOs: 263-285; and/or

CDR-H3: selected from any one of SEQ ID NOs: 286-316.

In an embodiment, the light chain variable region comprisescomplementarity determining regions CDR-L1 and/or CDR-L2 having thenucleic acid sequences set forth below:

CDR-L1: SEQ ID NO: 227 and/or

CDR-L2: SEQ ID NO: 228.

Variants of the CDRs that bind the different epitopes are described. Inaddition, the degeneracy of the genetic code allows for differentnucleic acids to encode the same amino acid sequence. Accordingly, alsoincluded are nucleotide sequences that hybridize to the nucleic acidsequences encoding the antibody and/or binding fragment thereofdisclosed herein under at least moderately stringent hybridizationconditions and which encode an antibody that also specifically bindsαKlotho polypeptide.

Also included in another embodiment are codon degenerate or optimizedsequences. In another embodiment, the nucleic acid sequences have atleast 70%, most preferably at least 80%, even more preferably at least90% and even most preferably at least 95% sequence identity to nucleicacid sequences encoding SEQ ID NOs: 227-316 (as shown in Table 3B and3C).

The antibodies described herein can comprise one or more of the featuresdescribed herein.

In an embodiment, the nucleic acid is an isolated nucleic acid.

Another aspect is a vector comprising the nucleic acid herein disclosed.In an embodiment, the vector is an isolated vector.

The vector can be any vector suitable for producing an antibody and/orbinding fragment thereof, including for example vectors describedherein. Possible expression vectors include but are not limited tocosmids, plasmids, or modified viruses (e.g. replication defectiveretroviruses, adenoviruses and adeno-associated viruses).

A further aspect is a recombinant cell producing the antibody and/orbinding fragment thereof herein disclosed or the vector hereindisclosed.

The recombinant cell can be generated using any cell suitable forproducing a polypeptide, for example suitable for producing an antibodyand/or binding fragment thereof.

Suitable host cells include a wide variety of prokaryotic and eukaryotichost cells. For example, the proteins of the invention may be expressedin bacterial cells such as E. coli, insect cells (using baculovirus),yeast cells or mammalian cells.

More particularly, bacterial host cells suitable for producingrecombinant antibody producing cells include E. coli, B. subtilis,Salmonella typhimurium, and various species within the genusPseudomonas, Streptomyces, and Staphylococcus, as well as many otherbacterial species well known to one of ordinary skill in the art.Suitable bacterial expression vectors preferably comprise a promoterwhich functions in the host cell, one or more selectable phenotypicmarkers, and a bacterial origin of replication. Representative promotersinclude the β-lactamase (penicillinase) and lactose promoter system, thetrp promoter and the tac promoter. Representative selectable markersinclude various antibiotic resistance markers such as the kanamycin orampicillin resistance genes. Suitable expression vectors include but arenot limited to bacteriophages such as lambda derivatives or plasmidssuch as pBR322, the pUC plasmids pUC18, pUC19, pUC118, pUC119, andpNH8A, pNH16a, pNH18a, and Bluescript M13 (Stratagene, La Jolla,Calif.).

Suitable yeast and fungi host cells include, but are not limited toSaccharomyces cerevisiae, Schizosaccharomyces pombe, the genera Pichiaor Kluyveromyces and various species of the genus Aspergillus. Examplesof vectors for expression in yeast S. cerivisiae include pYepSec1, pMFa,pJRY88, and pYES2 (Invitrogen Corporation, San Diego, Calif.). Protocolsfor the transformation of yeast and fungi are well known to those ofordinary skill in the art.

Suitable mammalian cells include, among others: COS (e.g., ATCC No. CRL1650 or 1651), BHK (e.g. ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa(e.g., ATCC No. CCL 2), 293 (ATCC No. 1573), NS-1 cells and anyderivatives of these lines.

In an embodiment, the mammalian cells used to produce a recombinantantibody are selected from CHO, HEK293 cells or Freestyle™ 293-F cells(Life technologies). FreeStyle 293-F cell line is derived from the 293cell line and can be used with the FreeStyle™ MAX 293 Expression System,FreeStyle™ 293 Expression System or other expression systems.

Suitable expression vectors for directing expression in mammalian cellsgenerally include a promoter (e.g., derived from viral material such aspolyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40), as well asother transcriptional and translational control sequences.

In an embodiment, the vector is designed for production of light chainor IgG1 heavy chain.

Suitable insect cells include cells and cell lines from Bombyx orSpodotera species. Baculovirus vectors available for expression ofproteins in cultured insect cells (SF 9 cells) include the pAc seriesand the pVL series.

The recombinant expression vectors may also contain genes which encode afusion moiety (i.e. a “fusion protein”) which provides increasedexpression or stability of the recombinant peptide; increased solubilityof the recombinant peptide; and aid in the purification of the targetrecombinant peptide by acting as a ligand in affinity purification,including for example tags and labels described herein. Further, aproteolytic cleavage site may be added to the target recombinant proteinto allow separation of the recombinant protein from the fusion moietysubsequent to purification of the fusion protein. Typical fusionexpression vectors include pGEX (Amrad Corp., Melbourne, Australia),pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the recombinant protein.

“Operatively linked” is intended to mean that the nucleic acid is linkedto regulatory sequences in a manner which allows expression of thenucleic acid. Suitable regulatory sequences may be derived from avariety of sources, including bacterial, fungal, viral, mammalian, orinsect genes. Selection of appropriate regulatory sequences is dependenton the host cell chosen and may be readily accomplished by one ofordinary skill in the art. Examples of such regulatory sequencesinclude: a transcriptional promoter and enhancer or RNA polymerasebinding sequence, a ribosomal binding sequence, including a translationinitiation signal. Additionally, depending on the host cell chosen andthe vector employed, other sequences, such as an origin of replication,additional DNA restriction sites, enhancers, and sequences conferringinducibility of transcription may be incorporated into the expressionvector.

In an embodiment, expression of the antibody or binding fragment thereofis under the control of an inducible promoter. Examples of induciblenon-fusion expression vectors include pTrc (28) and pET 11d.

The recombinant expression vectors may also contain a marker gene whichfacilitates the selection of host cells transformed or transfected witha recombinant molecule of the invention. Examples of selectable markergenes are genes encoding a protein such as G418 and hygromycin whichconfer resistance to certain drugs, β-galactosidase, chloramphenicolacetyltransferase, firefly luciferase, or an immunoglobulin or portionthereof such as the Fc portion of an immunoglobulin preferably IgG.Transcription of the selectable marker gene is monitored by changes inthe concentration of the selectable marker protein such asβ-galactosidase, chloramphenicol acetyltransferase, or fireflyluciferase. If the selectable marker gene encodes a protein conferringantibiotic resistance such as neomycin resistance transformant cells canbe selected with G418. Cells that have incorporated the selectablemarker gene will survive, while the other cells die. This makes itpossible to visualize and assay for expression of recombinant expressionvectors of the invention and in particular to determine the effect of amutation on expression and phenotype. It will be appreciated thatselectable markers can be introduced on a separate vector from thenucleic acid of interest. Other selectable markers include fluorescentproteins such as GFP which may be cotransduced with the nucleic acid ofinterest.

Yet another aspect is a composition comprising the antibody and/orbinding fragment thereof, the nucleic acid herein disclosed or therecombinant cell herein disclosed, optionally in combination with asuitable diluent or carrier.

The composition can be a lyophilized powder or aqueous or non-aqueoussolution or suspensions, which may further contain antioxidants,buffers, bacteriostats and solutes. Other components that may be presentin such compositions include water, surfactants (such as Tween),alcohols, polyols, glycerin and vegetable oils, for example.

Suitable diluents for nucleic acids include but are not limited towater, saline solutions and ethanol.

Suitable diluents for polypeptides, including antibodies or fragmentsthereof and/or cells include but are not limited to saline solutions, pHbuffered solutions and glycerol solutions or other solutions suitablefor freezing polypeptides and/or cells.

The composition can further comprise stabilizing agents, for examplereducing agents, hydrophobic additives, and protease inhibitors whichare added to physiological buffers.

III. Methods

Another aspect of the disclosure is a method for isolating or producingan antibody and/or binding fragment thereof described herein withspecific binding affinity to αKlotho and that binds an epitope ofαKlotho that is different from the epitope recognized by the sb106antibody.

As previously mentioned, the sb106 antibody comprises a light chainvariable region comprising CDR-L1, CDR-L2 and CDR-L3 having amino acidsequences SEQ ID NO: 9, 10 and 5, respectively, and a heavy chainvariable region comprising CDR-H1, CDR-H2 and CDR-H3 having amino acidsequences SEQ ID NO: 6, 7 and 8, respectively.

Additional αKlotho specific antibodies that bind different epitopes aredesirable for example to establish detection assays. As described hereinantibodies to αKlotho were identified by antibody-phase displayselections performed on the extracellular domain (ECD) of human αKlothowhile in the presence of saturating levels of the original sb106 Fab.Using this method, 31 antibodies were identified as recognizing adistinct epitope of αKlotho and as binding αKlotho in the presence andabsence of sb106 (Example 10 and FIG. 9). The antibodies exhibit bindingaffinities (K_(D)) less than 10 nM, as shown in Table 5. In oneembodiment, the method comprises screening an antibody library for anantibody that binds soluble αKlotho polypeptide and isolating theantibody from an antibody library. For example, the antibody library canbe an antibody phage-display library. In one embodiment, the antibodyphage-display library is a human Fab phage-display library.

In an embodiment, αKlotho polypeptide is used to isolate an antibodythat specifically binds αKlotho polypeptide from the antibody library.In an embodiment, αKlotho complexed with an antibody or binding fragmentthat has CDR regions specific for epitope A, B or C is used to isolatean antibody that specifically binds αKlotho polypeptide from theantibody library.

In another embodiment, the isolated and purified antibody and/or bindingfragment thereof is affinity matured. Affinity maturation can beperformed as described for the initial selection, with antigen adsorbedto plastic plates, using a for example a phage library comprisingvariants of the CDR sequences.

A person skilled in the art will appreciate that several methods can beused to isolate and produce antibodies and/or binding fragments thereofwith specific binding affinity to folded αKlotho. A method that can beused is a phage display method. For example, a binary αKlotho-FGF1Rccomplex is produced in order to isolate and characterize the antibodyand/or binding fragment thereof. Phage from a human Fab phage-displayedlibrary are selected following several rounds of panning. Phage withspecific binding affinity to the binary αKlotho-FGF1 Rc complex, asdetermined by ELISA, are sequenced and cloned into vectors designed forproduction of light chain or heavy chain. The heavy chain can be forexample an IgG, or an IgG isotype such as an IgG1 or an IgG4. Antigenbinding fragments and IgG polypeptides are then affinity purified byusing, for example, Protein A affinity columns.

In another embodiment, a nucleic acid encoding an antibody describedherein is expressed in a host cell to make the antibody and/or bindingfragment thereof. In an embodiment, the method comprises:

-   -   a. expressing in a host cell a nucleic acid encoding an antibody        and/or binding fragment thereof herein disclosed;    -   b. culturing the host cell to produce the antibody and/or        binding fragment thereof; and    -   c. isolating and/or purifying the antibody and/or binding        fragment thereof from the host cell.

In some embodiments, a nucleic acid encoding a single chain antibody isexpressed. In other embodiments, multiple nucleic acids are expressed,for example encoding a nucleic acid, encoding an antibody light chainand a nucleic acid encoding an antibody heavy chain.

Suitable host cells and vectors are described above. Vectors and nucleicacids encoding an antibody described herein may be introduced intomammalian cells via conventional techniques such as calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran mediated transfection,lipofectin and other liposome based transfection agents, electroporationor microinjection.

Nucleic acid encoding an antibody described herein may be directlyintroduced into mammalian cells using delivery vehicles such asretroviral vectors, adenoviral vectors and DNA virus vectors.

As described in Example 13, the antibodies were tested using animmunoprecipitation-immunoblot assay as Fabs on human urine samples. Theresults demonstrate (FIG. 13) that the antibodies have the ability toimmunoprecipitate αKlotho. The antibodies were assayed for their abilityto immunoprecipitate αKlotho from human patient urine samples as Fabs.

V. Assays

The αKlotho specific antibodies disclosed herein bind different epitopesand can be used in a variety of assays for binding, detecting andmeasuring αKlotho in a sample. For example, the antibodies can be usedin a proximity ligation assay (PLA) as well as immunoprecipitationoptionally combined with immunoblot detection. The antibody baseddetection can also be combined with a mass spectrophometric assay, forexample as in the case of a particle-based flow cytometric assay.

Immunodetection methods as described herein generally involve thedetection or measuring of antibody:αKlotho complexes using antibodiesand/or binding fragments thereof disclosed herein. The detection of suchcomplexes is well known in the art and may be achieved through differentmethods, for example by using a detectable label or marker, such as aradioactive, fluorescent or enzymatic tag. Detection of these complexesmay also involve the use of a ligand such as a secondary antibody and/orbinding fragment thereof specific for αKlotho or for theantibody:αKlotho complex.

They can also be used to make detection assays, for example a sandwichELISA, as one antibody can be used as a capture reagent to isolate theαKlotho while another antibody binding a distinct epitope can be used asa detection reagent.

Epitopes A and B are located within amino acids 550-981 αKlotho andepitope C within amino acids 1-549 of αKlotho. As shown in Example 14,FIG. 14, three antibodies binding different epitopes were incubated withtwo different portions of αKlotho, amino acids 1-549 and amino acids34-981 of αKlotho. Clone 48 (sb106) which binds epitope A and clone 4808which binds epitope B both bind within amino acids 550-981 of αKlothowhereas clone 4831 which binds epitope C binds within amino acids 1-549.

Accordingly, another aspect is an immunoassay comprising one or moreantibodies and/or binding fragments thereof herein disclosed (e.g.specific for epitope B or C).

In an embodiment, the immunoassay is an enzyme linked immunosorbentassay (ELISA). Antibodies and/or binding fragments thereof may be usedin the context of detection assays such as ELISAs, for example sandwichELISAs. As shown in Example 14 and FIG. 15, the antibodies were used ascapture and detection antibodies for the detection of αKlotho insolution. Full length IgGs recognizing epitopes A (sb106), B (sb177) orC (sb200) were immobilized and incubated with αKlotho. The samples werethen incubated with biotinylated IgGs recognizing epitopes A, B or C.

In an embodiment, the ELISA is a sandwich ELISA comprising a captureantibody and a detection antibody, wherein the capture antibody is anantibody or binding fragment thereof that has CDRs identified herein andwhich specifically binds αKlotho, for example which specifically bindsepitope A, B or C and/or the detection antibody is an antibody orbinding fragment thereof that has CDRs identified herein and whichspecifically binds αKlotho, for example which specifically binds epitopeA, B or C, wherein the capture antibody and the detection antibody binddifferent epitopes.

In one embodiment, the capture antibody and the detection antibody areselected from an antibody and/or binding fragment thereof hereindisclosed, and the sb106 antibody having light and heavy chain variableregions comprising the amino acid sequences of SEQ ID NO: 11 and 12.

In another embodiment, one of the capture and detection antibodies is anantibody and/or binding fragment thereof herein disclosed having CDRsidentified in Table 3 and the other of the capture and detectionantibodies is an antibody with CDRs identified for sb106 antibody or avariant thereof, optionally having light and heavy chain variableregions comprising the amino acid sequences of SEQ ID NO: 11 and 12,respectively.

In one embodiment, the immunoassay comprises an antibody and/or bindingfragment thereof having CDRs described herein which binds epitope B or Cof αKlotho herein as well as an antibody with the CDRs of sb106 antibodyor a variant thereof identified as binding epitope A.

For example, the capture antibody binds epitope A and the detectionantibody binds epitope B. For example, the capture antibody bindsepitope A and the detection antibody binds epitope C. For example, thecapture antibody binds epitope B and the detection antibody bindsepitope A. For example, the capture antibody binds epitope B and thedetection antibody binds epitope C. For example, the capture antibodybinds epitope C and the detection antibody binds epitope A. For example,the capture antibody binds epitope C and the detection antibody bindsepitope B.

In one embodiment, the antibody or variant thereof is sb106-Fab(Fsb106).

In one embodiment, the immunoassay is for the detection and/or measuringof αKlotho polypeptide in a sample, wherein the method of making theimmunoassay comprises:

-   -   a) coating a solid support with the capture antibody;    -   b) contacting the capture antibody with the sample under        conditions to form a capture antibody:αKlotho complex;    -   c) removing unbound sample;    -   d) contacting the capture antibody:αKlotho complex with the        detection antibody;    -   e) removing unbound detection antibody; and    -   f) detecting and/or measuring the capture antibody:αKlotho        complex.

In an embodiment, the ELISA is a competitive ELISA. In an embodiment,the ELISA is a direct ELISA. In an embodiment, the ELISA is an indirectELISA.

As used herein, “solid supports” include any material to which αKlothopolypeptide and antibodies and/or binding fragments thereof hereindisclosed are capable of binding to. For example, the solid support caninclude plastic, glass, polystyrene, nylon, polypropylene, nylon,polyethylene, dextran, amylases, natural and modified celluloses andpolyacrylamides. For example, the solid support is a microtiter plate,magnetic beads, latex beads or array surfaces.

For example, the sample is contacted with an antibody and/or bindingfragment thereof under appropriate conditions, for example, at a giventemperature and for a sufficient period of time, to allow effectivebinding of αKlotho to the antibody, thus forming an antibody:αKlothocomplex, such as a capture antibody:αKlotho complex. For example, thecontacting step is carried out at room temperature for about 30 minutes,about 60 minutes, about 2 hours or about 4 hours. For example, thecontacting step is carried out at about 4° C. overnight.

For example, the antibody and/or binding fragment thereof disclosedherein is complexed with αKlotho in a suitable buffer. For example, thebuffer has a pH of about 5.0 to about 10.0. For example, the buffer hasa pH of 4.5, 6.5 or 7.4. For example, the buffer is a HBS-EP buffer, aKRH buffer or Tris-buffered saline. For example, the buffer comprisesBSA and/or Tween20.

For example, any unbound sample may be removed by washing so that onlythe formed antibody:αKlotho complex remains on the solid support. Forexample, the unbound sample is washed with phosphate-buffered saline,optionally comprising bovine serum albumin (BSA).

In an embodiment, the detection antibody is labelled and/or conjugatedto a tag.

For example, the detection antibody directly labelled and/or conjugated.For example, the detection antibody is indirectly labelled and/orconjugated. Indirect labels include for example fluorescent orchemiluminescent tags, metals, dyes or radionuclides attached to theantibody. Indirect labels include for example horseradish peroxidase,alkaline phosphatase (AP), beta-galactosidase and urease. For example,HRP can be used with a chromogenic substrate, for exampletetramethybenzidine, which produces a soluble product in the presence ofhydrogen peroxide that is detectable at 450 nm.

Yet another aspect is an assay for detecting and/or measuring level ofαKlotho polypeptide in a sample, the assay comprising:

-   -   a) contacting a sample with the antibody and/or binding fragment        thereof herein described under conditions to form an        antibody:αKlotho complex; and    -   b) detecting and/or measuring the antibody:αKlotho complex.

A further aspect is an assay for detecting and/or measuring solubleαKlotho the method comprising:

-   -   a) contacting a sample, the sample being a biological fluid,        with the antibody and/or binding fragment thereof herein        described under conditions to form an antibody:soluble αKlotho        complex; and    -   b) detecting and/or measuring the antibody:soluble αKlotho        complex.

In an embodiment, the assay is for detecting folded αKlotho and theassay is performed under non-denaturing or mildly denaturing conditions.

In an embodiment, the complex is detected directly for example whereinthe antibody is labeled with a detectable tag or fusion moiety. In anembodiment, the complex is detected indirectly using a secondaryantibody specific for the antibody:αKlotho complex.

In an embodiment, the assay is an immunoprecipitation, immunoblot,immunohistochemistry or immunocytochemistry proximity ligation assay(PLA), mass spectroscopy-based techniques and fluorescence-activatedcell sorting (FACS), proximity ligation assay (PLA), and massspectroscopy-based techniques.

In an embodiment, the method is for detecting soluble αKlotho, forexample wherein the sample is a biological fluid.

Detecting can be performed using methods that are qualitative ormeasured using quantitative methods, for example by comparing to astandard or standard curve.

In an embodiment, the biological fluid sample is blood, or a partthereof such as serum or plasma, or urine.

Yet another aspect relates to a method for screening, for diagnosing orfor detecting kidney insufficiency condition selected from chronickidney disease (CKD) and acute kidney injury (AKI) in a subject, themethod comprising:

-   -   a. measuring the level of αKlotho in a sample from a subject        optionally using an antibody or assay herein disclosed; and    -   b. comparing the level of αKlotho in the sample with a control,    -   wherein a decreased level of αKlotho in the sample compared to        the control is indicative that the subject has a kidney        condition selected from CKD or AKI.

In an embodiment, the control is a control value derived from a group ofsubjects without CKD or AKI e.g. normal controls.

In an embodiment, the CKD is early CKD, optionally stage 1, stage 2, orstage 3, stage 4, stage 5 or stage 6 CKD.

An additional aspect of the disclosure is a method for prognosing CKDprogression or AKI progression or lack thereof (e.g. recovery orworsening of disease), or extra-renal complication in CKD, which isassessed by measuring the level of αKlotho deficiency.

Accordingly an aspect is a method of prognosing a likelihood of recoveryafter AKI, the method comprising:

-   -   a. measuring a level of αKlotho in a sample from a subject; and    -   b. comparing the level of αKlotho in the sample with a control,        for example a control value derived from a group of subjects        that did not recover or progressed,    -   wherein an increased level of αKlotho in the sample compared to        the control is indicative that the subject has an increased        likelihood of recovery after AKI.

In an embodiment, the control is a control value derived from a group ofsubjects that did recover and a decreased level of αKlotho in the samplecompared to the control is indicative that the subject has a decreasedlikelihood of recovery after AKI and/or an increased likelihood ofdisease progression.

Another aspect is a method for prognosing a likelihood of long termcomplications after AKI, the method comprising:

-   -   a. measuring a level of αKlotho in a sample from a subject; and    -   b. comparing the level of αKlotho in the sample with a control,        for example a control value derived from a group of subjects        with long term complications or with increased number of long        term complications,    -   wherein an increased level of αKlotho in the sample compared to        the control is indicative that the subject has an increased        likelihood of having fewer long term complications after AKI.

In an embodiment, wherein the control is a control value derived from agroup of subjects without long term complications or with fewer longterm complications, a decreased level of αKlotho in the sample comparedto the control is indicative that the subject has an increasedlikelihood of having long term complications or an increased number oflong term complications after AKI.

A further aspect is a method for prognosing the likelihood ofprogression of CKD, the method comprising:

-   -   a. measuring a level of αKlotho in a sample from a subject; and    -   b. comparing the level of αKlotho in the sample with a control,    -   wherein an increased level of αKlotho in the sample compared to        the control, for example wherein the control is a control value        derived from a group of subjects that did not recover or        progressed is indicative that the subject has an increased        likelihood of recovering from CKD.

In an embodiment, the control is a control value derived from a group ofsubjects that did recover and a decreased level of αKlotho in the samplecompared to the control is indicative that the subject has a decreasedlikelihood of recovery after CKD and/or an increased likelihood ofdisease progression.

Yet another aspect is a method for prognosing extra-renal complicationsin CKD, the method comprising:

-   -   a. measuring a level of αKlotho in a sample from a subject; and    -   b. comparing the level of αKlotho in the sample with a control,    -   wherein an increased level of αKlotho in the sample compared to        the control is indicative that the subject has a higher        likelihood of having fewer extra-renal complications related to        CKD.

In an embodiment, wherein the control is a control value derived from agroup of subjects without extra-renal complications or with fewerextra-renal complications, an decreased level of αKlotho in the samplecompared to the control is indicative that the subject has a increasedlikelihood of having long term complications or an increased number ofextra-renal complications after CKD.

A further aspect is a method for monitoring a subject with a kidneyinsufficiency condition such as CKD or AKI, the method comprising:

-   -   a. measuring a level of αKlotho in a sample from a subject; and    -   b. comparing the level of αKlotho in the sample with a previous        reference sample other control,    -   wherein an increased level of αKlotho in the sample compared to        the previous reference sample or other control is indicative        that the subject has an ameliorating kidney condition and a        decreased level of αKlotho in the sample compared to the        previous reference sample or other control is indicative that        the subject has a worsening kidney condition.

The sample can for example be taken after the subject has received atreatment and compared for example to a pre-treatment sample.Alternatively the patient can be monitored after a repeating interval toassess for example if treatment or other intervention is necessary. Inan embodiment, the test is repeated and plotted to assess the subject'sprogression.

In an embodiment, the sample is a biological fluid such as blood, or afraction thereof such as plasma or serum and the method is for exampledetecting soluble αKlotho. In an embodiment the biological fluid isurine.

In another embodiment, the sample is selected from a fresh sample suchas a fresh biological fluid sample or tissue sample (e.g. including notfrozen or one time frozen (e.g. frozen a single time at the time ofobtaining the sample)) and a repeat frozen sample (e.g. frozen andthawed and frozen biological fluid sample or repeat frozen tissuesample). In an embodiment, the sample is a fixed sample such as a mildlyfixed sample wherein the fixation induces limited denaturation and/orunfolding.

In an embodiment, the level of αKlotho is measured using an antibody orbinding fragment described herein.

The methods disclosed herein to diagnose, detect or monitor a kidneydisease or prognose a kidney disease complication, can be used inaddition to or in combination with traditional diagnostic techniques forkidney disease.

Any antibody or combination of antibodies described herein or in theexamples can be used in the assays.

IV. Kits

A further aspect relates to a kit comprising i) an antibody and/orbinding fragment thereof, ii) a nucleic acid, iii) a composition or iv)a recombinant cell herein disclosed, comprised in a vial such as asterile vial or other housing and optionally a reference agent and/orinstructions for use thereof.

In an embodiment, the kit further comprises an additional antibodyand/or binding fragment thereof having a light chain variable regioncomprising CDR-L1, CDR-L2 and CDR-L3 having amino acid sequences SEQ IDNO: 9, 10 and 5, respectively, and a heavy chain variable regioncomprising CDR-H1, CDR-H2 and CDR-H3 having amino acid sequences SEQ IDNO: 6, 7 and 8, respectively. For example, the additional antibodyand/or binding fragment thereof is an sb106 antibody and/or bindingfragment thereof.

In an embodiment, the kit comprises components and/or is for use inperforming an assay described herein.

For example, the kit is an ELISA kit and can comprise a first antibody,e.g. a capture antibody, for example attached to a solid support, and asecond antibody, e.g. a detection antibody, that binds to αKlotho and/orthe capture antibody:αKlotho complex, and that is conjugated to adetectable label.

Any combination of antibodies described herein can be used.

In an embodiment, the kit is a diagnostic kit and the instructions aredirected to a method described herein.

The above disclosure generally describes the present application. A morecomplete understanding can be obtained by reference to the followingspecific examples. These examples are described solely for the purposeof illustration and are not intended to limit the scope of theapplication. Changes in form and substitution of equivalents arecontemplated as circumstances might suggest or render expedient.Although specific terms have been employed herein, such terms areintended in a descriptive sense and not for purposes of limitation.

The following non-limiting examples are illustrative of the presentdisclosure:

EXAMPLES Example 1

A synthetic antibody library was screened and an antigen-bindingfragment (Fab) with high affinity for human and rodent αKlotho wasgenerated. This novel antibody, sb106, was characterized usingrecombinant proteins, cultured cells, and body fluids and tissues fromhumans and rodents. αKlotho levels in serum and urine in human androdents can be accurately quantified, and it is demonstrated that bothserum and urine αKlotho are dramatically reduced in early human CKD. Thesb106 antibody is specific to the a-form of Klotho and is the firstknown one to successfully pull down αKlotho from patient serum samples,in a clean and specific manner, as compared to currently commerciallyavailable αKlotho detection reagents. In cells, it can immunoprecipitateαKlotho and label it by immunocytochemistry. In animals, the antibody isefficient at immunoprecipitating αKlotho from plasma. The ability of thesb106 antibody to detect small quantities of αKlotho from biologicalfluids makes it a valuable reagent for diagnosis of diseases where thelevel of αKlotho is abnormal. Moreover, sb106 antibody is valuable as aresearch reagent in studies of physiologic and pathologic states thatinvolve any FGF23-mediated signaling pathways. It can be used inspecific assays for soluble αKlotho in human and rodent samples such asserum using a variety of techniques such as enzyme-linked immunosorbentassay (ELISA), proximity ligation assay (PLA), and massspectroscopy-based techniques.

Example 2 Preparation of the Binary αKlotho-FGFR1c Complex

The ligand-binding domain of human FGFR1c (D142 to R365) was expressedin E. coli, refolded in vitro from inclusion bodies, and purified bypublished methods [72,73]. The extracellular domain of murine αKlotho(A35 to K982) was expressed in HEK293 cells with a C-terminal FLAG tag,and the binary complex of the αKlotho ectodomain and the FGFR1cligand-binding domain was prepared as described [9].

Isolation and Characterization of Sb106

Sb106 was isolated from a synthetic human Fab phage-displayed library(Library F) [74]. Binding selections, phage ELISAs and Fab proteinpurification were performed as described [67,75,76]. Briefly, phage fromlibrary F were cycled through rounds of panning with the binary complexof αKlotho extracellular domain and FGFR1c ligand-binding domain on96-well Maxisorp Immunoplates (Fisher Scientific, Nepean, ON, Canada) asthe capture target. After 5 rounds of selection, phage were producedfrom individual clones grown in a 96-well format and phage ELISAs wereperformed to detect specific binding clones. Clones that showed bindingwere subjected to DNA sequencing. A competitive binding ELISA wasperformed by pre-incubating sb106-phage with serial dilutions of solublehuman αKlotho (50-0.0005 nM×1 hour) prior to binding to an ELISA platecoated with human αKlotho. The genes encoding for variable heavy andlight chain domains of sb106 were cloned into vectors designed forproduction of light chain or IgG1 heavy chain, respectively, andsb106-IgG was expressed from 293F cells (Invivogen, San Diego, Calif.USA). Fab and IgG proteins were affinity-purified on Protein A affinitycolumns (GE Healthcare, Mississauga, ON, Canada).

Purification of Proteins

The binary complex of FGFR1c ligand-binding domain and murine αKlothoectodomain (referred to as αKlotho-FGFR1c complex) was prepared by apublished protocol [9]. The N-terminally hexahistidine tagged, matureform of human FGF23 (Y25 to I251) was expressed in E. coli and purifiedby published protocols [73,74,77].

Analysis of Fab Binding to αKlotho-FGFR1c Complex by SPR Spectroscopy

Real time protein-protein interactions were measured using a Biacore2000 surface plasmon resonance (SPR) spectrometer (Biacore AB/GEHealthcare) at 25° C. in HBS-EP buffer (10 mM HEPES-NaOH, pH 7.4, 150 mMNaCl, 3 mM EDTA, 0.005% (v/v) polysorbate 20). Proteins were covalentlycoupled through their free amino groups to carboxymethyl (CM) dextran ofresearch grade CM5 biosensor chips (Biacore AB/GE Healthcare). Proteinswere injected over a biosensor chip at a flow rate of 50 μl min-1, andat the end of each protein injection (180 s), HBS-EP buffer (50 μlmin⁻¹) was flowed over the chip to monitor dissociation for 180 s.Injecting 2.0 M NaCl in 10 mM sodium acetate, pH 4.5, or 10 mMsodium/potassium phosphate, pH 6.5 regenerated the chip surface inbetween protein injections. The data were processed with BiaEvaluationsoftware version 4.1 (Biacore AB/GE Healthcare). For each proteininjection, the non-specific SPR responses recorded for the control flowchannel were subtracted from the responses recorded for the sample flowchannel.

To examine whether Fabs selected by ELISA bind to the αKlotho-FGFR1ccomplex, the binary receptor complex was immobilized on a CM5 chip (˜42fmol mm⁻² of chip flow channel). To control for non-specific binding,bovine β-glucuronidase (Sigma-Aldrich), which is structurally related toeach of the two extracellular glycosidase-like domains of αKlotho, wascoupled to the control flow channel of the chip (˜45 fmol mm⁻² of flowchannel). 100 nM of each Fab were injected over the chip. As a control,binding of FGF23 to the immobilized αKlotho-FGFR1c complex was examined.

To test if the Fabs can compete with FGF23 and/or binding to theαKlotho-FGFR1c complex, FGF23 was immobilized on a CM5 chip (˜16 fmolmm⁻² of chip flow channel). FHF1B, which is structurally similar to FGFsbut has no FGFR binding [77], was used as control for non-specificbinding (˜15 fmol mm⁻² of control flow channel). 100 nM of Fab mixedwith 10 nM of αKlotho-FGFR1c complex (HBS-EP buffer) was injected overthe chip. For control, the binding competition was carried out withFGF23 as the competitor in solution.

Cell Culture, Animal and Human Studies

Cell lines: normal rat kidney (NRK) cells with native αKlotho expression(ATCC, Manassas, Va., USA), and HEK293 cells transfected with vectoronly, full-length transmembrane murine αKlotho, extracellular domain ofmurine αKlotho with C-terminal FLAG tag, or full-length murineβKlotho[78]. Cells were cultured at 37° C. in a 95% air, 5% CO₂atmosphere, passed in high-glucose (450 mg/dl) DMEM supplemented with10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100mg/ml).

Animal studies were approved by the University of Texas SouthwesternMedical Center Institutional Animal Care and Care Committee. All animalswere housed in the Animal Resource Center and experiments were performedin fully approved laboratories. Species used include: Sprague-Dawleyrats (Harlan. Indianapolis, Ind.), Klotho transgenic overexpressors(Tg-Klotho; EFmKL46 line)[79], homozygous αKlotho hypomorphic mice(KI/KI)[80], and their wild type littermates (129sv background).

Clinical history and routine lab data were obtained from electroniccharts. Blood samples from antecubital venipuncture were spun, and theserum was frozen at −80° C. Fresh urine was spun at 4,000 g and thesupernatant was frozen at −80° C.

Immunocytochemistry and Immunohistochemistry

HEK293 cells transfected with vector or the stated αKlotho plasmids andseeded (1.8×10⁵ cells/ml) on 12-well glass cover slips pre-treated withpoly-lysine and grown overnight. The cells were washed (4° C. PBS×3),fixed with 3% paraformaldehyde (4° C.×10 min), washed (ice cold PBS×3),blocked with 1% BSA (PBS 440° C.×10 min), incubated with sb106-Fab(5ug/ml in 1% BSA, PBS) washed (PBS 40° C.×5), incubated withanti-FLAG-Alexa488 (Cell Signaling; diluted 1:400 in PBS containing 1%BSA; 1 hour; 200C), washed (PBS; 4° C.×4), and then inverted onto glassslides containing a drop of antifade with DAPI (Invitrogen) and dried atroom temperature in the dark. After 24 hours, the slides were stored at−20° C. Images were obtained on a WaveFX spinning disc confocalmicroscope.

The parathyroid and thyroid (en bloc with the trachea) were from adultSprague Dawley rats. For non-fixed fresh parathyroids, tissues wereembedded in OCT medium and frozen with isopentane pre-cooled in liquidN₂ immediately. For fixed parathyroid samples, tissue was immersed in 4%paraformaldehyde in PBS pH 7.4 at 4° C. overnight, washed with PBS, andembedded in OCT medium and frozen with isopentane pre-cooled in liquidN₂. Four μm thick cryostat sections were made, washed in PBS (15 min),and permeabilized in 0.1% TritonX-100 (10 min). For labeling, sectionswere blocked (PBS, 1.5% BSA, 10% goat serum; 40 min) and incubated withthe primary antibody sb106 (21 μg/ml in blocking solution; 40Covernight). After washing (PBS), sections were incubated with the Alexa546 goat anti-human IgG (1:800 dilution, Invitrogen) for 1 hour at roomtemperature. After additional washes with PBS, the sections were fixedwith 4% paraformaldehyde in PBS, washed with PBS, and mounted andvisualized with a Zeiss LSM510 microscope.

Example 3: αKlotho Assays and Detailed Methods

The ELISA was performed as instructed by the manufacturer(Immuno-Biological Laboratory, Japan). For the IP-IB assay, typically 50μl of serum or urine were diluted with KRH buffer [25 mM Hepes-NaOH (pH7.4), 120 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 1.3 mM CaCl2, 1.3 mM KH2PO4]to a final volume of 0.5 ml incubated with 2 μg of sb106-Fab overnightat 4° C. in low binding, siliconized tubes. Sepharose beads (50 μL)conjugated with anti-FLAG antibody (50% v/v) prewashed 3× with KRHbuffer were added, incubated (4° C.×2 h), and washed (KRH-500 μl pertube ×3; 22° C.). The immune complex was eluted with 2×SDS sampleloading buffer (50 μl; 100° C.×3 min; 4° C.×3 min; spun), andfractionated by SDS-PAGE, transferred to nitrocellulose membranes andblotted with anti-KL1 antibody (KM2076, 1:4000 or 3.1 mg/mL, 1:10000dilution) and diluent (Dako#S3022, Carpinteria, Calif., USA) overnight(4° C., rocker). The membrane was washed (×3, Tris-buffered saline with0.1% Tween; TBS-T), exposed to anti-rat IgG2A (LSBio cat#LS-C59051,1:20000 in 5% milk/2% goat serum/TBS-T×1 h) and washed (×3 TBS-T). Forchemiluminescence, the membrane was covered with SuperSignal West FemtoMaximum Sensitivity substrate (Thermo Scientific, Rockford, Ill., USA)and exposed for 30-90 s. The 130-kD bands were scanned, and density wascompared with internal control samples of know amount of Klotho usingAdobe Photoshop CS4.

Example 4: Identification of an Anti-αKlotho Synthetic Fab

After rounds of biopanning of a phage-displayed synthetic Fab library onrecombinant αKlotho ectodomain complexed with the ligand-binding domainof fibroblast growth factor receptor (FGFR)1c, several binding phageswere identified. Clone sb106 (FIG. 1A) was chosen for furthercharacterization. In phage ELISA (FIG. 1B), sb106-phage bound to bothhuman and mouse αKlotho, demonstrating cross species reactivity, and toeither αKlotho alone or in complex with FGFR1c, indicating that itsepitope is not obscured by co-receptor complex formation. Sb106-phagedid not bind to FGFR1c alone, neutravidin (NAV) or bovine serum albumin(BSA). Sb106 binds to human αKlotho with affinity in the single-digitnanomolar range (IC50=1.7 nM, FIG. 1C). Sb106-Fab also binds with highaffinity to the binary αKlotho-FGFR1c complex immobilized on a biosensorchip (FIG. 7A) and it does not interfere with ternary complex formationbetween FGF23, αKlotho and FGFR1c (FIG. 7B).

Example 5: Characterization of the Anti-αKlotho Fab Sb106

Using the unique CDR sequences of sb106 (FIG. 1A), both Fab andfull-length IgG proteins were produced (e.g. Fab was produced inbacterial cells and IgG proteins in 293F cells). Sb106 was highlyreactive against αKlotho under native conditions. Immunoblot signalsunder denaturing conditions against mouse, rat, and human kidney tissuewere weak, but in samples from transgenic mice overexpressing αKlotho[79], sb106-Fab detected a band corresponding to the full-lengthextracellular domain of αKlotho (FIG. 2A). In cultured cells, sb106-Fabwas not able to detect αKlotho in immunoblots under denaturingconditions with lysates from NRK cells expressing native αKlotho but itwas able to detect overexpressed antigen in cell lysates from HEK293cells transfected with αKlotho (FIG. 2B). In immunohistochemistry withfreshly frozen unfixed rat parathyroid tissue (and other tissues knownto express αKlotho, sb106-IgG detected αKlotho but the same tissue wasnegative when fixed (FIG. 2C), suggesting that sb106 binds to thenatively folded FGFR1-αKlotho complex (FIG. 1B). In immunocytochemicalstains with freshly fixed cells, unequivocal staining was obtained inHEK293 cells heterologously overexpressing αKlotho but not in cellsoverexpressing βKlotho (FIG. 2D). The cells were seeded at 1.8×10⁵/ml on12-well glass cover slips treated with poly-lysine and grown overnight.Cells were washed 3 times with ice cold PBS, fixed for 10 min on ice (3%paraformaldehyde), washed 3 times with cold PBS, blocked for 10 min (1%BSA in cold PBS). Cells were then incubated with sb106-Fab (5 μg/ml in1% BSA in PBS) for 1 hr. Cells were washed 5 times with cold PBS (2 mineach) and then incubated with anti-FLAG-Alexa488 (the C-terminus of theFab light chain contains a Flag epitope tag) (1:400 in 1% BSA in PBS)for 1 hr, while being protected from light. Cells were washed 4 timeswith cold PBS (5 min each). The glass coverslips were then inverted ontoglass slides containing a drop of antifade reagent with DAPI. Imageswere collected on a spinning disc confocal microscope. Even in cellsoverexpressing αKlotho, prolonged fixation greatly diminished orabolished the staining with sb106. These data indicate that sb106 reactsspecifically with natively folded human, rat, and mouse αKlotho but notwith denatured αKlotho protein.

Example 6: Immunoprecipitation of αKlotho

The ability of sb106-Fab to precipitate soluble αKlotho was tested usinga sequential immunoprecipitation-immunoblot (IP-IB) assay. Sb106-Fabpulled down αKlotho from total cell lysates and conditioned cell culturemedium and from αKlotho-overexpressing cells (FIG. 3A). The sb106-Fabpull-down was compared with that of an anti-FLAG antibody using solubleαKlotho with a C-terminal FLAG tag in HEK293 cells. Sb106-Fab andanti-FLAG precipitated proteins with the exact same electrophoreticmobilities.

Sb106-Fab precipitated a ˜130 kDa protein from human, mouse, and ratsera that reacted with the anti-αKlotho antibody KM2076 (FIG. 3B). IPfrom urine also showed a ˜130 kDa band (FIG. 3B), whereas the post-IPurine samples did not show such a band in immunoblot. To further supportthe authenticity of the IP-IB band by sb106, the intensity of this bandwas examined in human sera from a normal individual vs. a patient withCKD stage 5, and sera from a wild type mouse vs. a homozygous αKlothohypomorph (FIG. 3C). Only the ˜130 kDa band (FIG. 3C) was reduced inhuman advanced CKD and was absent in the αKlotho-deficient mice(kl/kl)[80].

Example 7: αKlotho Levels in Human CKD

The IP-IB method was tested to determine whether it can reliablydetermine serum αKlotho levels from a single center database of CKDpatients. Recombinant human αKlotho was spiked in known amounts to testthe linearity of the assay as well as the extrapolated y-intercept.IP-IB was performed with sera from a normal healthy volunteer and apatient with stage 5 CKD spiked with a range of different concentrationsof recombinant αKlotho (FIG. 4A). There was graded increase in signalwith the incrementally inoculated exogenous αKlotho. The serum from theCKD patient also showed increases in signal with increasing exogenousαKlotho but, at any given concentration of αKlotho, the signal intensitywas lower than the normal serum.

Interestingly, the serum from the healthy volunteer gave the same signalin the absence or presence of a protease inhibitor cocktail, whereas theserum from the CKD patient displayed a marked increase in measuredαKlotho levels in the presence of a protease inhibitor (FIG. 4B). Thesefindings suggest that while endogenous αKlotho exists in a stable steadystate in uremia, the exogenously added αKlotho may undergo proteolysisin uremic serum but not in normal serum. A quantitative summary of thespiking experiment is shown in FIG. 4C. Both healthy and CKD sera showedlinear responses to αKlotho inoculation but the signal from CKD sera hasa lower slope. When protease inhibitors were included, the slope of theCKD line approached that of the healthy subject without affecting itsintercept. Extrapolation to zero inoculation showed that the serum fromthe normal individual had 31.1 μM αKlotho while that from the CKDpatient had 8.5 μM αKlotho. Similar extrapolations were obtained from anumber of other subjects with normal renal function or with CKD.

The nature of patients recruited from the CKD clinic resembles thenational profile of CKD where diabetes and hypertension predominate(Table 1). Despite the scatter, there is a clear progressive decline ofαKlotho with stages of CKD (FIG. 5A). The decrease in serum αKlothooccurred early in CKD, and preceded high FGF23, high PTH, andhyperphosphatemia (Table 1). The IP-IB assay was directly compared witha commercial αKlotho ELISA kit with the same samples (FIG. 5B). Overall,there is a correlation between the two but there is separation on bothsides of the line of identity. In fresh samples, the ELISA shows highervalues than IP-IB (grey diamonds to the left of the line of identity,FIG. 5B) but in samples that have been through one or more cycles offreeze-thaw, the ELISA values are much lower (black diamonds to theright of the line of identity, FIG. 5B). When the exact same sampleswere tested by the two methods before and after repeated freeze-thaw,the IP-IB assay gave more stable results while the ELISA values droppedrapidly (FIG. 5C).

Low urinary αKlotho in human CKD patients by directly immunoblottingurine was previously described[12]. The IP-IB assay with sb106-Fabshowed dramatic reduction of urinary αKlotho in CKD patients (FIG. 6A).In contradistinction from serum, the ELISA yielded more comparablevalues to the IP-IB assay in the urine but the magnitude of decrease inαKlotho concentration is more dramatic when detected by the IP-IB assaythan by ELISA (FIG. 6B). These results show that human CKD is a state ofαKlotho deficiency in both serum and urine.

Hence using an immunoprecipitation-immunoblot (IP-IB) assay, both theserum and urinary levels of full-length soluble αKlotho was measured inhumans and it was established that human CKD is associated with αKlothodeficiency in serum and urine. αKlotho levels were detectably lower inearly CKD preceding disturbances in other parameters of mineralmetabolism and levels progressively declined with CKD stages.Exogenously added αKlotho is inherently unstable in the CKD milieu.

Antibody-based reagents are valuable tools in both research and clinicalsettings for detection of proteins, protein isolation and purification,and numerous downstream applications. The commercial reagents availablefor αKlotho detection are limiting; for example there are no antibodiesfor specifically detecting natively folded αKlotho protein. Moreover,the commercial ELISA kit for αKlotho detection yields highly variableresults.

Synthetic antibodies with designed antigen-binding sites can befine-tuned and tailored for molecular recognition of vast repertoires oftargets. Coupled with in vitro phage-display, selections are performedin the absence of tolerance mechanisms that eliminate self-reactiveantibodies. Selections with an antibody library yielded sb106, anantibody with specificity for natively folded human, mouse and ratαKlotho.

In addition to its role in mineral metabolism, soluble αKlothocirculates in many bodily fluids and has multiple “house-keeping”functions that maintain cellular integrity throughout the body. Althoughthe mechanism of action of soluble αKlotho remains poorly understood,the biologic impact of αKlotho deficiency is unequivocally shown [81].αKlotho transcripts are present in multiple organs but the kidney by farhas the highest expression [80]. CKD is a state of multiple metabolicderangements and is a complex syndrome from accumulation ofunder-excreted endogenous and exogenous toxins as well as deficiency insubstances responsible for health maintenance.

There is evidence in experimental animals that both AKI and CKD arestates of systemic αKlotho deficiency. Not only is this an early andsensitive biomarker, restoration of αKlotho can ameliorate the renaldysfunction. Independent from its renoprotective effects, αKlotho canalso reduce the extrarenal complications in CKD [12,82]. Based on thepreclinical data, anti-αKlotho antibody may have both diagnostic andprognostic value.

Validation of the IP-IB Assay and Comparison with the Commercial ELISA

Available commercial assays for αKlotho have no consistent correlationbetween them [46, 83]. Studies in healthy humans and CKD patients basedon one ELISA [58] have yielded contradictory results. The absolutelevels of αKlotho in normal and CKD ranged from 0.4[47] to over 2000μg/ml [41] with most readings in the mid to high hundreds [48, 50, 55,58-60,83]. Based on this assay, αKlotho levels have been described to below [48, 52, 54, 57-60], no relationship to [40, 41, 50, 51, 53] or evenincreased [44, 47] with decreasing glomerular filtration rate (GFR).Likewise, αKlotho levels have been reported as not changed or decreasingwith age [42, 53, 58, 59]. This renders the interpretation of humanαKlotho data nearly impossible, and the collective data derived fromdifferent centers will have no value.

A high affinity synthetic antibody that recognizes αKlotho in itsnatively folded conformation (FIGS. 1-3) was generated. Sb106-Fab or IgGpulls down αKlotho from cell lysate, culture medium, serum, and urine.Additional bands may be shorter fragments of αKlotho but the intensityof these bands did not decrease in the kl/kl mice arguing against thispossibility. The ˜130 kDa band has been analyzed which is full lengthsoluble αKlotho; something that the ELISA cannot achieve.

The linearity of the spiking experiment indicates that all theinoculated αKlotho is detected (FIG. 4). An unexpected finding was thatexogenously added recombinant αKlotho is proteolytically degraded inuremic serum whereas no such phenomenon was observed in normal sera.This challenges the view that the low αKlotho in kidney disease stemssolely from decreased production and opens up additional mechanisms andnew avenues for investigation. In addition to uncovering new mechanismsof αKlotho deficiency in CKD, this may have significant implications interms of recombinant αKlotho replacement.

There is graded reduction in serum αKlotho with advancing CKD (FIG. 5A).The coefficient of variation of the IP-IB assay was 4% for serum and 7%for urine. The IP-IB assay also showed very low urinary αKlotho inadvanced CKD (FIG. 6). In fact, the reduction in urinary αKlotho is moredramatic than that in serum and may represent a more sensitive markerfor CKD.

Both IP-IB and the commercial ELISA detected the low urine αKlotho inCKD, although the absolute levels of αKlotho are much higher with theELISA assay and the percent reduction is not the same as with the IP-IBassay. With drastic reduction in urinary αKlotho levels in CKD, the twoassays yielded the same conclusion with quantitative differences. Thesituation in serum is different. Although there is overall positivecorrelation, the comparison of the two assays completely segregated intotwo groups (FIG. 5B). The fresh samples showed higher readings for theELISA while the stored samples yielded extremely low results with theELISA. One possibility is that the ELISA is measuring αKlotho and someother reacting proteins in fresh samples. While the IP-IB assay did losesome efficacy with repeated freeze-thaw, this is a much more seriousproblem with the ELISA.

Another advantage of the IP-IB assay is that it can measure αKlotho inboth humans and rodents equally well, whereas the use of the currentlyavailable ELISA in rodent can potentially be problematic as it detectsvery high circulating αKlotho levels in rats with CKD which is a stateof pan-αKlotho deficiency. [68]

Example 8

Additional CDR sequences are provided in Table 2. Homologous mutationswere introduced at each amino acid position, meaning that for eachposition either the original amino acid was retained or the closest“homolog” to that amino acid (e.g. conservative amino acid change) wasintroduced and a new Fab-phage library was constructed. Selections wereperformed using the new library using the alphaKlotho-FGFR1c complex asan antigen. Clones that bound to the antigen were isolated and sequencedand are shown in Table 2. The binding affinity is expected to be similaror better than Sb106.

Example 9 Human Studies

Nine human subjects (49.066.2 years) who underwent right heartcatheterization were enrolled for this study. During right heartcatheterization, suprarenal and infrarenal vena caval blood samples wereobtained and sera were immediately separated after centrifugation at 4°C. and stored at −80° C. for future study. Serum αKlotho was determinedby immunoprecipitation-immunoblot assay described herein. Briefly, 0.1ml serum was immunoprecipitated with a synthetic anti-αKlotho Fab(sb106) and immune complex was eluted with Laemmli sample buffer, andsubject to immunoblot with KM2076 antibody. The specific signals on theautoradiograms based on 130 kD mobility were quantified with ImageJProgram (National Institutes of Health (NIH), Bethesda, Md.).

Animal Studies

αKlotho hypomorphic (kl/kl) mice, kl/kl mice and their wild-type (WT)littermates were maintained at the Animal Research Center at theUniversity of Texas Southwestern Medical Center. Currently all mice are129 S1/SVImJ (129 SV) background age from 6 to 8 weeks. NormalSprague-Dawley (SD) rats (220-250 g body weight) were purchased fromCharles River Laboratories (Wilmington, Mass.). For αKlotho clearancestudy, rats underwent bilateral nephrectomy (anephric rats) orlaparotomy with manual manipulation of the kidneys (sham rats). Rats ormice were intravenously or intraperitoneally injected once with labeledfull extracellular domain of recombinant mouse αKlotho protein (rMKI)(R&D Systems, Minneapolis, Minn.) at a dose of 0.1 mg/kg body weight. Toexamine if secretases modulate blood αKlotho, doxycline hyclate(Sigma-Aldrich, St. Louis, Mo.), an a-secretase inhibitor at 25mg/kg/day, and/or 3-secretase inhibitor III (Calbiochem, Billerica,Mass.) at 2.5 mg/kg/day were intraperitoneally injected into normal WTmice daily for 2 days, blood and kidneys were harvested at 48 hours todetermine serum and renal αKlotho.

Antibodies

Rat monoclonal anti-human Klotho antibody, KM20761,2 was used forimmunoblotting and immunoelectron microscopy; and the syntheticanti-αKlotho antibody sb10663 was used for immunoprecipitation of serumKlotho.

Clearance of Labeled αKlotho in Rats and Mice

Normal Munich Wistar rats (220-250 g BW) were anesthetized with Inactin(100 mg/kg BW), and a bonus of labeled αKlotho was injected through thejugular vein (0.1 mg/kg BW). For the experiment of injection ofI251-labeled αKlotho or I251-labeled albumin, fluid collection byfree-flow micropuncture of Bowman's space, and proximal convolutedtubules was performed using published methods. In brief, the left kidneywas exposed, and the left ureter was catheterized for urine collection.Proximal tubules were identified by their characteristic configurationafter lissamine green dye injection and punctured with glasscapillaries. The volume of fluid was measured in a calibratedconstant-bore glass capillary. The radioactivity of fluids wasdetermined by scintillation accounting and normalized to fluid volume.At specified time points, blood was drawn from retro-orbital venoussinus, and spot urine was collected. I251-labeled αKlotho orI251-labeled albumin in collected urine and serum was quantified byscintillation counting. Homogenates of different organs were made andradioactivity in organ homogenates was measured by scintillationcounting, and normalized to protein in organ homogenates. Organ sections(10 mm) were subjected to autoradiography.

Immunoelectron Microscopy

Mouse recombinant Klotho protein (0.1 mg/kg BW) was intraperitoneallyinjected once into kl/kl mice and mice were sacrificed 24 hours afterinjection. Kidneys were harvested and fixed with 2.5% paraformaldehydevia aortic perfusion, removed, and post-fixed in 4% paraformaldehyde (4°C. for 4 hours). Immunogold labeling of ultrathin frozen tissue sectionswas performed as described.21 Kidney cortex was infiltrated with 2.3 Msucrose overnight, frozen in liquid nitrogen, and 70-80-nm-thicksections were made (Ultramicrotome Reichert Ultracut E; LeicaMicrosystems, Wetzlar, Germany) and mounted on Formvar-coated nickelgrids. The sections were incubated with KM2076 antibody and followed byincubation with gold conjugated protein A (10-nm gold particles,Sigma-Aldrich) for 60 minutes. After staining with uranyl acetate,sections were visualized with Jeol 1200 EX transmission electronmicroscope (Jeol Ltd., Akishima, Japan).

Results

The role of the kidney in circulating αKlotho production and handlingwas examined. Serum levels of αKlotho protein in suprarenal andinfrarenal vena cava of normal rats by direct puncture and humansubjects who underwent right heart catheterization. All patients hadeGFR≥60 ml/min/1.73 m². Similar infrarenal-to-suprarenal increment incaval αKlotho level was observed in both rat and human serum samples.Serum αKlotho levels were plotted against serum erythropoietin, awell-known renal-derived hormone, and it was found that as serumerythropoietin rose, and serum creatinine (SCr) decreased frominfrarenal-to-suprarenal inferior vena cava, whereas αKlotho increasedindicating that the kidney secretes αKlotho into the circulation.

When both kidneys were removed from rats, serum αKlotho level droppedsignificantly to about half the normal level in one day. The anephricstate did not permit studies to continue for longer than 40-50 hours.

The method of αKlotho clearance from circulation was investigated. Thelevels of circulating exogenous αKlotho protein in anephric rats weresimilar to those in normal rats immediately after injection, but thehalf-life of exogenous αKlotho protein in normal rats was much shorterthan that in anephric rats and the half-life of endogenous αKlotho uponnephrectomy closely approximates that of exogenous αKlotho in theanephric rats. Further experiments examining the anatomic fate ofintravenous injected exogenous labeled αKlotho supported that the kidneymay be a major organ of αKlotho uptake as well as its excretion.

Injected labeled αKlotho protein was prominently distributed in thekidney and spleen, sparsely in the heart, and not detectable in aorta,brain, and muscle. Further experiments tracking clearance ofradioactively labelled exogenous αKlotho in serum and urine, supportedthat αKlotho protein is cleared from blood through the kidney to theurine.

Based on these and further experiments, it was determined that the (1)the kidney produces and releases soluble αKlotho into the systemiccirculation by secretases-mediated shedding of the ectodomain ofαKlotho, (2) the kidney is an important organ to clear soluble αKlothofrom the circulation, (3) αKlotho traffics across renal tubules frombasolateral to intracellular location and is then secreted across theapical membrane into the urinary lumen.

Example 10: Identification of Additional Antibodies that Bind αKlotho

The original antibody reagent to αKlotho, sb106 (clone 48), as well asCDR variants derived from sb106, all bind to a single common epitope. Toestablish a detection assay, one needs antibodies with differentepitopes, as one surface/epitope is used to isolate or capture theprotein while a second, distinct epitope is needed for the detectionreagent. Thus another selection campaign was undertaken to identifyantibodies which bind to epitopes different and distinct from that ofsb106. These antibodies to αKlotho were identified by antibody-phagedisplay selections performed on the extracellular domain (ECD) of humanαKlotho while in the presence of saturating levels of sb106 Fab (50ug/ml). The protein purchased from R&D Systems (5334-KL) covers aminoacids E34-S981 with a 6-His tag attached at the C-terminus.

In total, 31 new antibodies from a synthetic antibody library (Perssonet al, 2013 J Mol Biol) were identified and shown to bind αKlotho, inthe presence and absence of the first αKlotho antibody, sb106 (FIG. 9).CDRs of these clones are shown in Table 3 (amino acid sequence in 3A andnucleotide sequences in 3B & 3C) and examples of full-length sequencesin Table 4. The clones were first given a name, and then ID numbers.Both are shown here for reference.

All sequences were sub-cloned into the Fab expression vector (RH2.2),expressed and purified. As was with the phage, all Fab clones bound tothe target antigen by ELISA (FIG. 10).

Example 11: Epitope Grouping

There are at least 2 new epitopes on αKlotho within this set ofadditional antibodies. Epitope grouping experiments were performed usinga competition ELISA strategy. Given the large number of clones withinthis set, preliminary studies revealed 2 distinct epitopes separate fromthe epitope that sb106 (48) binds. Representatives of these epitopes(4804 and 4819) were then used alongside sb106 (48) for assessment ofeach of the clones within the set (FIG. 11). Where sb106 (48) representsepitope A, 23 of the new clones were grouped into epitope B, and 4clones were found for epitope C (FIG. 11 and summarized in Table 5). For3 clones (4806, 4810 and 4817) no determination was made due to problemswith the phage. Clone 4828 did not show competition with any of the 3represented epitopes.

Example 12: Affinity Estimates

Affinity estimates for each of the antibodies were determined by surfaceplasmon resonance (SPR). Fabs were immobilized using an anti-H+Lantibody and serial dilutions of αKlotho were injected. Binding curvesare shown in FIG. 12 and the binding affinities (KDs) are summarized inTable 5. The affinities range from 240 μM to 8.7 nM.

Example 13: Immunoprecipitation

The antibodies were assayed for their ability to immunoprecipitateαKlotho from human patient urine samples as Fabs (FIG. 13). 50 ul ofhuman urine samples from normal volunteers where incubated within 400 ulKRH buffer with 1 ug/ml of Fab (sb173-203, sb106) overnight at 4° C. 50ul of M2 anti-FLAG Sepharose beads were then added, and incubated for 2hrs at 4° C. (the C-terminus of the Fab light chains contains a FLAGepitope tag). The beads were washed 3 times with 500 ul KRH buffer, andbound proteins were eluted with 2×LDS sample loading buffer containing100 mM DTT. Immunoblot was performed with a commercial primary ratanti-Klotho antibody (KM2076) followed by a standard anti-rat IgGsecondary for visualization. Super Signal West Femto substrate was usedas the chemiluminescent substrate. More than half of the clones behavedas well or better than sb106.

Example 14: ELISAs

Select clones, representing the new epitopes (4808 for B, and 4831 forC) were subcloned into full length IgGs, expressed and purified andfurther characterized alongside sb106 (48). Full length IgGs wereimmobilized at 1 ug/ml (capture), blocked with buffer containing 1% BSA,then incubated with either 20 ng of biotinylated αKlotho (aa34-981), 20ng of biotinylated αKlotho (aal-549) in capture buffer (Tris bufferedsaline pH 7.4+0.1% BSA+0.05% Tween20) or capture buffer only (BSA) atroom temperature for 1 hour. After washing with PBS+0.05% Tween20, thecaptured biotinylated αKlotho was detected using an HRP-streptavidinreagent. Colorimetric HRP reagents allow for absorbance readings at 450nm. All three IgGs predictably captured αKlotho from solution (FIG. 14),however only 4831 was able to detect a truncated version of ECD ofαKlotho, further characterizing the difference in epitope recognitionfrom sb106.

Finally, a matrix of ELISA experiments were performed such that each ofthe 3 distinct epitope clones were used as capture and detectionantibodies pairwise which each other for the detection of αKlotho insolution (FIG. 15). Full length IgGs were immobilized at 1 ug/ml(capture), blocked with PBS+5% BSA, then incubated with 5 nM of αKlothoat room temperature for 1 hour. After washing with PBS+0.05% Tween20,the samples where then incubated with biotinylated preps of the sameIgGs (dectrion, *b) for 30 minutes at room temperature. After washingagain, bound IgGs were detected using an HRP-streptavidin reagent.Colorimetric HRP reagents allow for absorbance readings at 450 nm.Duplicate data points are shown. Grey highlights self against self.These experiments show that the 3 epitopes the antibodies bind aresuitably compatible with each other for the desired purpose of using ina diagnostic kit.

Example 15: Cross-Reactivity to Human and Mouse αKlotho

Fab ELISA using both human and mouse αKlotho was carried out forassessing cross-reactivity to human and mouse αKlotho (FIG. 16). Fabs (5ug/ml) were incubated with immobilized αKlotho human, mouse, orneutravidin (NA) and SBA as negative controls. After washing off unboundFab, bound Fab were detected using an HRP-conjugated anti-Flag antibody(Fabs have Falg tag on the end of their light chains). Colorimetric HRPreagents allow for absorbance readings at 450 nm.

Estimated affinities against human and mouse antigen for select αKlothoFabs were obtained by surface plasmon resonance on a ProteOn XPR36 (FIG.17). Fabs were captured using an anti-IgG(H+L) antibody and serialdilutions of αKlotho were injected. Binding curves were fitted to theLangmuir model. (A) Determined Kon, Koff and KD values. (B) and (C)Binding curves for human and mouse antigen respectively.

Example 16: Fab Binding to αKlotho-FGFR1c Complex

Fab proteins (clones 4804-4824, 4826-4834) were adsorbed to an ELISAplate (Maxisorb) at a concentration of 15 ug/ml, for 1 hour at roomtemperature, and then blocked with PBS+0.5% BSA for 1 hour at roomtemperature. The plate was washed with PBS+0.05% Tween 20 and thenincubated with 2ug/ml of the following proteins: αKlotho (Fc dimer),complex of αKlotho-FGFR1c (Fc complex), Fc only, or PBS, at roomtemperature for 1 hour. Unbound antigen was washed away, with 6 washesof PBS+0.05% Tween 20, and then the wells were incubated with agoat-anti-mouse (mG2a, Jackson ImmunoResearch Laboratories) for 30minutes at room temperature. Unbound anti-mouse antibody was washedaway, with 6 washes of PBS+0.05% Tween 20, and then the wells wereincubated with an anti-goat-HRP reagent for 30 minutes at roomtemperature. Unbound anti-goat-HRP antibody was washed away, with 6washes of PBS+0.05% Tween 20, and then colourmetric HRP reagents (TMBsubstrate and stop solution) were used and absorbance was read at 450nm.

The results show that the αKlotho antibodies (clones 4804-4824,4826-4834) bind αKlotho alone and in complex (αKlotho-FGFR1c). sb106(clone 48) was used as a control for binding to both αKlotho alone andin complex (αKlotho-FGFR1c).

TABLE 1 Characteristics of human subjects Serum Gender PCr Serum Pi HCO₃⁻ Serum PTH FGF23 25(OH)Vitamin D Etiology of CKD Subject n Age (M/F)(mg/dl) (mg/dl) (mM) (pg/ml) (pg/ml) (ng/ml) (number subjects*) Healthy34 50 ± 17 14/20 0.8 ± 0.2  3.6 ± 0.6 23 ± 2 59 ± 25 30 ± 10   32 ± 10None CKD1 10 43 ± 10 7/3 0.8 ± 0.1  3.9 ± 0.5 25 ± 2 47 ± 19 61 ± 23  26± 7 DM (1) HTN (3) GN (7) CKD2 11 50 ± 22 4/7 1.1 ± 0.2  3.6 ± 0.5 26 ±2 56 ± 22 70 ± 27   21 ± 13 DM (2), HTN (4), GN (4), RK (3) CKD3 10 57 ±17 5/5 1.7 ± 0.4^(#) 3.2 ± 0.8 25 ± 3 86 ± 51 79 ± 18^(#) 25 ± 8 DM (3),HTN (7), GN (3), IN (1) CKD4 14 62 ± 13 8/6 2.7 ± 0.6^(#) 3.5 ± 0.9 24 ±3  202 ± 101^(#) 204 ± 173^(#) 21 ± 8 DM (4), HTN (10), GN (3), RK (1)CKD5 11 62 ± 12 5/6 4.7 ± 2.0^(#)  5.1 ± 3.5^(#) 21 ± 3  223 ± 188^(#)580 ± 427^(#)  21 ± 9^(#) DM (7), HTN (7), GN (2) Dialysis 14 50 ± 126/8 11.9 ± 15.6^(#)  4.8 ± 1.7^(#) 22 ± 5  500 ± 650^(#) 760 ± 286^(#)26 ± 8 DM (7), HTN (10), GN (5), PKD (1) n = number of subjects; PCr =plasma creatinien; GFR = estimated glomerular filtration rate; Serum Pi= serum phosphorus; Serum HCO3− = serum bicarbonate PTH = parathyroidhormone; FGF23+ Fibroblast growth factor 23; DM = Diabetes mellitus; HTN= hypertension; GN = glomerulonephritis; RK = Remnant kidney; IN =Interstitial nephritis; PKD = Polycystic kidney disease. *Some patientscarry more than one diagnosis. Results are shown as mean ± standarddeviation. ^(#)p < 0.05 compared to healthy volunteers. ANOVA

TABLE 2 CDR sequence variations (Sequences shown correspond to positions which are variable in the selection library.Sequences listed under column L3 refer to CDR-L3 but omit the  first and second amino terminal amino acid residues and the carboxy terminal amino acid residue of IMGT CDR-L3. Sequenceslisted under column H1 refer to CDR-H1 but omit thefirst 3 amino terminal amino acid residues of IMGT CDR-H1and the carboxy terminal amino acid residue thereofcorresponds to IMGT VH domain position 39. Sequences listed under column H2 correspond to IMGT VH domain positions55-66, which includes CDR-H2 and framework region residues at positions 55 and 66. Sequences listed under column H3refer to IMGT CDR-H3, but are lacking the first two amino terminal and the last two carboxy terminal amino acids of IMGTCDR-H3. IMGT CDR amino acid residues per se are underlined. Variable FR amino acid residues are not underlined.) L3 ID H1 ID H2 IDsb106 A G Y S P I 5 I S Y Y S I 6 Y I S P S Y G Y T S 7 E12 A G Y A P I15 I A Y Y A V 16 F I A P S Y G Y S S 17 E2 A G F A P I 19 I S Y F S V20 F I S P A Y G F T A 21 C12 A A F A P V 23 I A F Y A V 24 F V S A S YG Y T S 25 E3 S A F S P V 27 V S F Y S I 28 Y V A P A Y G Y S A 29 D7 AA F S P V 31 V S F F S I 32 Y I S P S Y G Y S S 33 E4 A G F A P I 35 I SY Y A V 36 F V S P A Y G F S S 37 E6 A A F A P V 39 F S S S S I 40 F V SP A Y G F T A 41 E10 A G Y A P V 43 I S Y Y S I 44 Y I S P S F G Y T A45 C9 A A F A P V 47 I S Y Y S I 48 F I A P A F G Y S S 49 Dll A A F S PI 51 I A Y F S I 52 Y V S P A Y G Y T S 53 El A G Y A P I 55 I A F Y S I56 Y V S P A Y A Y T A 57 D5 A G F A P I 59 V S F Y S I 60 S I S S S Y GY T Y 61 D12 A G F A P V 63 V S F F S I 64 S I S S S Y G Y T Y 65 E8 S AY A P V 67 V A F Y S I 68 F I A P S Y G Y S A 69 Ell A G F A P V 71 I AF F S I 72 F V S P A Y G Y T A 73 Fl S A Y S P V 75 I A F Y S I 76 F V SP A Y A Y S A 77 D1 A A F A P V 79 F S S S S I 80 Y I S P A Y G Y S A 81C10 V S Y Y S I 83 S I S S S Y G Y T S 84 D6 S A F S P V 86 I A F F A V87 F V S P S F G Y S S 88 D8 A A F A P V 90 V A F Y S I 91 Y I S P A Y AY S A 92 C11 I S Y F S V 94 Y V S P S F A F S S 95 F2 A A Y A P V 97 V AF Y S V 98 S I S S S Y G Y T A 99 D9 A A Y A P V 101 I A Y Y A V 102 F VS P A Y G F T S 103 E5 A A F A P V 105 I A F Y A V 106 Y V A P P Y A Y SA 107 D10 A A Y A P V 109 V S F Y S I 110 Y I S P A Y G Y T S 111 D3 S AY S P V 113 V A Y Y S I 114 Y I S P A F G Y S S 115 D4 A A Y A P I 117 VA F Y A V 118 S I S S S Y G Y T Y 119 H3 ID sb106 Y Y V Y A S H G W A GY G M 8 E12 F Y V Y A S N A W A G Y G M 18 E2 F Y V Y A A N G W A G Y GM 22 C12 F Y V Y A A N G W A G Y G M 26 E3 F Y V Y A A H G W A G Y G M30 D7 F Y V Y A A N G W A G Y G M 34 E4 F F V Y A A H G W A G Y G M 38E6 F F V Y S S H G W A G Y G M 42 E10 F Y V Y S S H G W A G Y G M 46 C9F Y V Y A A N G W A G Y G M 50 D11 F Y V Y S A N G W A G Y G M 54 El F YV Y A A H G W A G Y G M 58 D5 F Y V Y A S N G W A G Y G M 62 D12 F Y V YS S H G W A G Y G M 66 E8 F F V Y A A H G W A G Y G M 70 Ell F Y V Y S AN G W A G Y G M 74 Fl F Y V Y S A N G W A G Y G M 78 D1 F F V Y S A N AW S G Y G M 82 C10 F F V Y A A H G W A G Y G M 85 D6 F Y V Y A A H G W AG Y G M 89 D8 Y F V Y A S N G W A G Y G M 93 C11 F F V Y S A H G W A G YG M 96 F2 F Y V V A A H G W A G Y G M 100 D9 F Y V V S S H G W A G F G M104 E5 F Y V V S A H G W A G Y G M 108 D10 F Y V V S A H G W A G Y G M112 D3 F Y V V A A N G W A G Y G M 116 D4 Y Y V V A A H G W A G Y G M120

TABLE 2A SEQ ID NO: 1 X₁X₂X₃X₄PX₅wherein X₁ is A or S, X₂ is G or A, X₃ is Y or F, X₄ is S or A, X₅ is I or VSEQ ID NO: 2 X₆X₇X₈X₉X₁₀X₁₁wherein X₆ is I or V, X₇ is S or A, X₈ is Y, F or S, X₉ is Y, F or S, X₁₀ is S or A andX₁₁ is I or V SEQ ID NO: 3 X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁wherein X₁₂ is Y, F or S, X₁₃ is I or V, X₁₄ is S or A, X₁₅ is P or S, X₁₆ is S or A,X₁₇ is Y or F, X₁₈ is G or A, X₁₉ is Y or F, X₂₀ is T or Sand X₂₁ is S, A or YSEQ ID NO: 4 X₂₂X₂₃VYX₂₄X₂₅X₂₆X₂₇WX₂₈GX₂₉GMwherein X₂₂ is Y or F, X₂₃ is Y or F, X₂₄ is A or S, X₂₅ is S or A, X₂₆ is H or N, X₂₇is G or A, X₂₈ is A or S and X₂₉ is Y or F

TABLE 3A CDR amino acid sequences for additional aKlotho antibodies (The carboxy terminal amino acid residue ofthe sequences listed under column CDR-H1 corresponds to IMGT VH domain position 39; and the amino terminal residueand the carboxy terminal residue of the sequences listed under column CDR-H2 correspond to IMGT VH domain positions55 and 66, respectively. Amino acid residues of IMGT CDRs are underlined, amino acid residues of IMGT framework regionsare not underlined, and residues at IMGT positions which were randomized in the selection library are bold.) clone SEQ CDRSEQ SEQ  SEQ  SEQ  SEQ Epit- ID ID ID ID ID ID ID name ome CDR-L1 NO: L2NO: CDR-L3 NO: CDR-H1 NO: CDR-H2 NO: CDR-H3 NO: 4804 sb173 B QSVSSA 140SAS 141 QQSSYSLIT 142 GFNLYSYS I 157 Y ISSSSGST Y 175 ARGWGGGYWFYPVYGIDY196 4805 sb174 g QSVSSA 140 SAS 141 QQSSGWYHFLFT 143 GFNLSYYS M 158 SISSYYGST Y 176 ARGGGYYSGPYAGFDY 197 4806 sb175 ND QSVSSA 140 SAS 141QQSSYSLIT 142 GFNISSSS I 159 Y ISSSYSST S 177 ARSSGGGYYHWWVVPYAMDY 1984807 sb176 g QSVSSA 140 SAS 141 QQSSYSLIT 142 GFNLSSSY M 160 S IYPSYGSTS 178 ARGVVPSYYYWFWPYGAIDY 199 4808 sb177 g QSVSSA 140 SAS 141 QQSSYSLIT142 GFNFSSSS I 161 S ISSSYGYT Y 179 ARPYSAYYWAWYGPGGALDY 200 4809 sb178g QSVSSA 140 SAS 141 QQSGWAYHPIT 144 GFNIYSYY I 162 S IYSYYGST S 180ARSGPWAWYGLDY 201 4810 sb179 ND QSVSSA 140 SAS 141 QQSSYSLIT 142GFNLSSSS I 163 Y ISPSYGST S 181 ARSGYYSGAYWHWWVVPYAMDY 202 4811 sb180 gQSVSSA 140 SAS 141 QQGYALFT 145 GFNLYYSY M 164 S IYSSSSYT S 182ARSPSWWVSYHSALDY 203 4812 sb181 g QSVSSA 140 SAS 141 QQGYWLFT 146GFNLSSYS M 165 S ISSYSGYT S 183 ARSYSWWWSVSYAMDY 204 4813 sb182 g QSVSSA140 SAS 141 QQAAWGGAPIT 147 GFNLYSSS I 166 S ISPYSGYT Y 184ARYYSGWYSPAWWYGIDY 205 4814 sb183 C QSVSSA 140 SAS 141 QQSSPPIT 148GFNLYSYS M 167 S ISPYSGYT Y 184 ARSFFPYSYWVYGGGMDY 206 4815 sb184 gQSVSSA 140 SAS 141 QQSSYSLIT 142 GFNFSSSS I 161 S ISSSYGYT Y 179ARGFSSSAHWYWSWYGPGGGFDY 207 4816 sb185 g QSVSSA 140 SAS 141 QQSSYSLIT142 GFNFSSSS I 161 S ISSSYGYT Y 179 ARGWYAAYSVYWFGGHASYGLDY 208 4817sb186 ND QSVSSA 140 SAS 141 QQSSYSLIT 142 GFNFSSSS I 161 S ISSSYGYT Y179 ARGYPSSGAAWFWFSHPGSAMDY 209 4818 sb187 g QSVSSA 140 SAS 141 QQPYSPIT149 GFNISYSS I 168 S ISPYSGYT Y 184 ARSGHSVYWWWSHFGMDY 210 4819 sb188 CQSVSSA 140 SAS 141 QQGSYYWWSPIT 150 GFNIYSYS M 169 S IYPSSSYT Y 185ARAGYFSAYYSSWGAMDY 211 4820 sb189 g QSVSSA 140 SAS 141 QQSPWGAYLIT 151GFNISSYY M 170 S IYSSYSST Y 186 ARGAWAMDY 212 4821 sb190 g QSVSSA 140SAS 141 QQSSYSLIT 142 GFNLYYSY M 164 S ISPYSGST Y 187 ARSGFSSWWWVVSYAFDY213 4822 sb191 g QSVSSA 140 SAS 141 QQSSYSLIT 142 GFNFSSSS I 161 SISSSYGYT Y 179 ARAGWYSSWWWSAWGAGGGLDY 214 4823 sb192 g QSVSSA 140 SAS141 QQSSYSLIT 142 GFNFSSSS I 161 Y IYSSYGYT Y 188 ARAAHYGYYVHSGLDY 2154824 sb193 g QSVSSA 140 SAS 141 QQSSYSLIT 142 GFNLSSSS I 163 S ISSSYGYTY 179 ARHGYGYFFWGYYGPGSAMDY 216 4825 sb194 g QSVSSA 140 SAS 141QQSSYSLIT 142 GFNLYSSS I 166 S ISPYYSYT Y 189 ARSVYSWYWSSWGPGSALDY 2174826 sb195 g QSVSSA 140 SAS 141 QQAGFFSYPIT 152 GFNISSYY M 170 SISSSYGYT Y 179 ARGYPASSYYYPSSALDY 218 4827 sb196 g QSVSSA 140 SAS 141QQSSYSLIT 142 GFNFSSSS I 161 Y ISSSSGST S 190 ARAYHSYFYGSYWSYGWAGALDY219 4828 sb197 ND QSVSSA 140 SAS 141 QQSSYSLIT 142 GFNFSSSS I 161 SISSSYGYT Y 179 ARYVVGGWWYHYGMDY 220 4829 sb198 g QSVSSA 140 SAS 141QQGGGLIT 153 GFNISSYY I 171 S IYSSYGYT S 191 ARYSWSPYWWWAYSGLDY 221 4830sb199 C QSVSSA 140 SAS 141 QQYSWYWYSPIT 154 GFNIYYSS I 172 S IYPYYSYT Y192 ARSVASALDY 222 4831 sb200 C QSVSSA 140 SAS 141 QQYSYYYASPIT 155GFNIYSSS I 173 S IYPYSGYT Y 193 ARYSWGSSFWPGYGFDY 223 4832 sb201 BQSVSSA 140 SAS 141 QQSSYSLIT 142 GFNFSSSS I 161 Y ISSSYGYT S 194ARASGWFSHFYPAAVSGMDY 224 4833 sb202 B QSVSSA 140 SAS 141 QQSSHGHYPIT 156GFNLSSYY M 174 S IYPSYSST Y 195 ARSSYSVYFWWYVSAMDY 225 4834 sb203 BQSVSSA 140 SAS 141 QQSSYSLIT 142 GFNFSSSS I 161 S ISSSYGYT Y 179ARAVSFYYWAWYGPGFAMDY 226

TABLE 3BLight chain CDR nucleotide sequences for additional αKlotho antibodiesSEQ  SEQ  SEQ  ID ID ID clone ID name Epitome CDR-L1 NO: CDR-L2 NO:CDR-L3 NO: 4804 sb173 B CAGTCCGTGTCCAGCGCT 227 TCGGCATCC 228CAGCAATCTTCTTATTCTCTGATCACG 229 4805 sb174 B CAGTCCGTGTCCAGCGCT 227TCGGCATCC 228 CAGCAATCTTCTGGTTGGTACCATTTCCTGTTCACG 230 4806 sb175 NDCAGTCCGTGTCCAGCGCT 227 TCGGCATCC 228 CAGCAATCTTCTTATTCTCTGATCACG 2294807 sb176 B CAGTCCGTGTCCAGCGCT 227 TCGGCATCC 228CAGCAATCTTCTTATTCTCTGATCACG 229 4808 sb177 B CAGTCCGTGTCCAGCGCT 227TCGGCATCC 228 CAGCAATCTTCTTATTCTCTGATCACG 229 4809 sb178 BCAGTCCGTGTCCAGCGCT 227 TCGGCATCC 228 CAGCAATCTGGTTGGGCTTACCATCCGATCACG231 4810 sb179 ND CAGTCCGTGTCCAGCGCT 227 TCGGCATCC 228CAGCAATCTTCTTATTCTCTGATCACG 229 4811 sb180 B CAGTCCGTGTCCAGCGCT 227TCGGCATCC 228 CAGCAAGGTTACGCTCTGTTCACG 232 4812 sb181 BCAGTCCGTGTCCAGCGCT 227 TCGGCATCC 228 CAGCAAGGTTACTGGCTGTTCACG 233 4813sb182 B CAGTCCGTGTCCAGCGCT 227 TCGGCATCC 228CAGCAAGCTGCTTGGGGTGGTGCTCCGATCACG 234 4814 sb183 C CAGTCCGTGTCCAGCGCT227 TCGGCATCC 228 CAGCAATCTTCTCCGCCGATCACG 235 4815 sb184 BCAGTCCGTGTCCAGCGCT 227 TCGGCATCC 228 CAGCAATCTTCTTATTCTCTGATCACG 2294816 sb185 B CAGTCCGTGTCCAGCGCT 227 TCGGCATCC 228CAGCAATCTTCTTATTCTCTGATCACG 229 4817 sb186 ND CAGTCCGTGTCCAGCGCT 227TCGGCATCC 228 CAGCAATCTTCTTATTCTCTGATCACG 229 4818 sb187 BCAGTCCGTGTCCAGCGCT 227 TCGGCATCC 228 CAGCAACCGTACTCTCCGATCACG 236 4819sb188 C CAGTCCGTGTCCAGCGCT 227 TCGGCATCC 228CAGCAAGGTTCTTACTACTGGTGGTCTCCGATCACG 237 4820 sb189 B CAGTCCGTGTCCAGCGCT227 TCGGCATCC 228 CAGCAATCTCCGTGGGGTGCTTACCTGATCACG 238 4821 sb190 BCAGTCCGTGTCCAGCGCT 227 TCGGCATCC 228 CAGCAATCTTCTTATTCTCTGATCACG 2294822 sb191 B CAGTCCGTGTCCAGCGCT 227 TCGGCATCC 228CAGCAATCTTCTTATTCTCTGATCACG 229 4823 sb192 B CAGTCCGTGTCCAGCGCT 227TCGGCATCC 228 CAGCAATCTTCTTATTCTCTGATCACG 229 4824 sb193 BCAGTCCGTGTCCAGCGCT 227 TCGGCATCC 228 CAGCAATCTTCTTATTCTCTGATCACG 2294825 sb194 B CAGTCCGTGTCCAGCGCT 227 TCGGCATCC 228CAGCAATCTTCTTATTCTCTGATCACG 229 4826 sb195 B CAGTCCGTGTCCAGCGCT 227TCGGCATCC 228 CAGCAAGCTGGTTTCTTCTCTTACCCGATCACG 239 4827 sb196 BCAGTCCGTGTCCAGCGCT 227 TCGGCATCC 228 CAGCAATCTTCTTATTCTCTGATCACG 2294828 sb197 ND CAGTCCGTGTCCAGCGCT 227 TCGGCATCC 228CAGCAATCTTCTTATTCTCTGATCACG 229 4829 sb198 B CAGTCCGTGTCCAGCGCT 227TCGGCATCC 228 CAGCAAGGTGGTGGTCTGATCACG 240 4830 sb199 CCAGTCCGTGTCCAGCGCT 227 TCGGCATCC 228CAGCAATACTCTTGGTACTGGTACTCTCCGATCACG 241 4831 sb200 C CAGTCCGTGTCCAGCGCT227 TCGGCATCC 228 CAGCAATACTCTTACTACTACGCTTCTCCGATCACG 242 4832 sb201 BCAGTCCGTGTCCAGCGCT 227 TCGGCATCC 228 CAGCAATCTTCTTATTCTCTGATCACG 2294833 sb202 B CAGTCCGTGTCCAGCGCT 227 TCGGCATCC 228CAGCAATCTTCTCATGGTCATTACCCGATCACG 243 4834 sb203 B CAGTCCGTGTCCAGCGCT227 TCGGCATCC 228 CAGCAATCTTCTTATTCTCTGATCACG 229

TABLE 3CHeavy chain CDR nucleotide sequences for additional αKlotho antibodies (sequences encoding IMGTCDR residues are underlined, sequences encoding IMGT framework region residues are not underlined)SEQ SEQ SEQ clone ID ID ID ID name Epitome CDR-H1 NO: CDR-H2 NO: CDR-H3NO: 4804 sb173 B GGCTTCAACCTCTATTCTTATTC 244TATATTTCTTCTTCTTCTGGCTCTACTTAT 263GCTCGCGGTTGGGGTGGTGGTTACTGGTTCTACCCGGTTTACGGTATT 286 TATC GACTAC 4805sb174 B GGCTTCAACCTCTCTTATTATTC 245 TCTATTTCTTCTTATTATGGCTCTACTTAT 264GCTCGCGGTGGTGGTTACTACTCTGGTCCGTACGCTGGTTTTGACTAC 287 TATG 4806 sb175 NDGGCTTCAACATCTCTTCTTCTTC 246 TATATTTCTTCTTCTTATAGCTCTACTTCT 265GCTCGCTCTTCTGGTGGTGGTTACTACCATTGGTGGGTTGTTCCGTAC 288 TATC GCTATGGACTAC4807 sb176 B GGCTTCAACCTCTCTTCTTCTTA 247 TCTATTTATCCTTCTTATGGCTCTACTTCT266 GCTCGCGGTGTTGTTCCGTCTTACTACTACTGGTTCTGGCCGTACGGT 289 TATGGCTATTGACTAC 4808 sb177 B GGCTTCAACTTTTCTTCTTCTTC 248TCTATTTCTTCTTCTTATGGCTATACTTAT 267GCTCGCCCGTACTCTGCTTACTACTGGGCTTGGTACGGTCCGGGTGGT 290 TATA GCTTTGGACTAC4809 sb178 B GGCTTCAACATCTATTCTTATTA 249 TCTATTTATTCTTATTATGGCTCTACTTCT268 GCTCGCTCTGGTCCGTGGGCTTGGTACGGTTTGGACTAC 291 TATC 4810 sb179 NDGGCTTCAACCTCTCTTCTTCTTC 250 TATATTTCTCCTTCTTATGGCTCTACTTCT 269GCTCGCTCTGGTTACTACTCTGGTGCTTACTGGCATTGGTGGGTTGTTC 292 TATCCGTACGCTATGGACTAC 4811 sb180 B GGCTTCAACCTCTATTATTCTTA 251TCTATTTATTCTTCTTCTAGCTATACTTCT 270GCTCGCTCTCCGTCTTGGTGGGTTTCTTACCATTCTGCTTTGGACTAC 293 TATG 4812 sb181 BGGCTTCAACCTCTCTTCTTATTC 252 TCTATTTCTTCTTATTCTGGCTATACTTCT 271GCTCGCTCTTACTCTTGGTGGTGGTCTGTTTCTTACGCTATGGACTAC 294 TATG 4813 sb182 BGGCTTCAACCTCTATTCTTCTTC 253 TCTATCTCTCCTTATTCTGGCTATACTTAT 272GCTCGCTACTACTCTGGTTGGTACTCTCCGGCTTGGTGGTACGGTATT 295 TATC GACTAC 4814sb183 C GGCTTCAACCTCTATTCTTATTC 254 TCTATTTCTCCTTATTCTGGCTATACTTAT 273GCTCGCTCTTTCTTCCCGTACTCTTACTGGGTTTACGGTGGTGGTATGG 296 TATG ACTAC 4815sb184 B GGCTTCAACTTTTCTTCTTCTTC 248 TCTATTTCTTCTTCTTACGGTTACACTTAC 274GCTCGCGGTTTCTCTTCTTCTGCTCATTGGTACTGGTCTTGGTACGGTC 297 TATACGGGTGGTGGTTTTGACTAC 4816 sb185 B GGCTTCAACTTTTCTTCTTCTTC 248TCTATTTCTTCTTCTTATGGCTATACTTAT 267GCTCGCGGTTGGTACGCTGCTTACTCTGTTTACTGGTTCGGTGGTCAT 298 TATAGCTTCTTACGGTTTGGACTAC 4817 sb186 ND GGCTTCAACTTTTCTTCTTCTTC 248TCTATTTCTTCTTCTTATGGCTATACTTAT 267GCTCGCGGTTACCCGTCTTCTGGTGCTGCTTGGTTCTGGTTCTCTCATC 299 TATACGGGTTCTGCTATGGACTAC 4818 sb187 B GGCTTCAACATTTCTTACTCTTC 255TCTATTTCTCCTTATTCTGGCTATACTTAT 273GCTCGCTCTGGTCATTCTGTTTACTGGTGGTGGTCTCATTTCGGTATGG 300 TATT ACTAC 4819sb188 C GGCTTCAACATCTATTCTTATTC 256 TCTATTTATCCTTCTTCTAGCTATACTTAT 275GCTCGCGCTGGTTACTTCTCTGCTTACTACTCTTCTTGGGGTGCTATGG 301 TATG ACTAC 4820sb189 B GGCTTCAACATCTCTTCTTATTA 257 TCTATTTATTCTTCTTATAGCTCTACTTAT 276GCTCGCGGTGCTTGGGCTATGGACTAC 302 TATG 4821 sb190 BGGCTTCAACCTCTATTATTCTTA 251 TCTATTTCTCCTTATTCTGGCTCTACTTAT 277GCTCGCTCTGGTTTCTCTTCTTGGTGGTGGGTTGTTTCTTACGCTTTTG 303 TATG ACTAC 4822sb191 B GGCTTCAACTTTTCTTCTTCTTC 248 TCTATTTCTTCTTCTTATGGCTATACTTAT 267GCTCGCGCTGGTTGGTACTCTTCTTGGTGGTGGTCTGCTTGGGGTGCT 304 TATAGGTGGTGGTTTGGACTAC 4823 sb192 B GGCTTCAACTTTTCTTCTTCTTC 248TATATTTATTCTTCTTATGGCTATACTTAT 278GCTCGCGCTGCTCATTACGGTTACTACGTTCATTCTGGTTTGGACTAC 305 TATA 4824 sb193 BGGCTTCAACCTCTCTTCTTCTTC 258 TCTATTTCTTCTTCTTATGGCTATACTTAT 267GCTCGCCATGGTTACGGTTACTTCTTCTGGGGTTACTACGGTCCGGGT 306 TATATCTGCTATGGACTAC 4825 sb194 B GGCTTCAACCTCTATTCTTCTTC 253TCTATTTCTCCTTATTATAGCTATACTTAT 279GCTCGCTCTGTTTACTCTTGGTACTGGTCTTCTTGGGGTCCGGGTTCTG 307 TATC CTTTGGACTAC4826 sb195 B GGCTTCAACATCTCTTCTTATTA 257 TCTATTTCTTCTTCTTATGGCTATACTTAT267 GCTCGCGGTTACCCGGCTTCTTCTTACTACTACCCGTCTTCTGCTTTGG 308 TATG ACTAC4827 sb196 B GGCTTCAACTTTTCTTCTTCTTC 248 TATATTTCTTCTTCTTCTGGCTCTACTTCT280 GCTCGCGCTTACCATTCTTACTTCTACGGTTCTTACTGGTCTTACGGTT 309 TATAGGGCTGGTGCTTTGGACTAC 4828 sb197 ND GGCTTCAACTTTTCTTCTTCTTC 248TCTATTTCTTCTTCTTATGGCTATACTTAT 267GCTCGCTACGTTGTTGGTGGTTGGTGGTACCATTACGGTATGGACTAC 310 TATA 4829 sb198 BGGCTTCAACATCTCTTCTTATTA 259 TCTATTTATTCTTCTTATGGCTATACTTCT 281GCTCGCTACTCTTGGTCTCCGTACTGGTGGTGGGCTTACTCTGGTTTG 311 TATC GACTAC 4830sb199 C GGCTTCAACATCTATTATTCTTC 260 TCTATTTATCCTTATTATAGCTATACTTAT 282GCTCGCTCTGTTGCTTCTGCTTTGGACTAC 312 TATC 4831 sb200 CGGCTTCAACATCTATTCTTCTTC 261 TCTATTTATCCTTATTCTGGCTATACTTAT 283GCTCGCTACTCTTGGGGTTCTTCTTTCTGGCCGGGTTACGGTTTTGACT 313 TATC AC 4832 sb201B GGCTTCAACTTTTCTTCTTCTTC 248 TATATTTCTTCTTCTTATGGCTATACTTCT 284GCTCGCGCTTCTGGTTGGTTCTCTCATTTCTACCCGGCTGCTGTTTCTG 314 TATA GTATGGACTAC4833 sb202 B GGCTTCAACCTCTCTTCTTATTA 262 TCTATTTATCCTTCTTATAGCTCTACTTAT285 GCTCGCTCTTCTTACTCTGTTTACTTCTGGTGGTACGTTTCTGCTATGG 315 TATG ACTAC4834 sb203 B GGCTTCAACTTTTCTTCTTCTTC 248 TCTATTTCTTCTTCTTATGGCTATACTTAT267 GCTCGCGCTGTTTCTTTCTACTACTGGGCTTGGTACGGTCCGGGTTTC 316 TATAGCTATGGACTAC

TABLE 3DLight chain CDR-L3 amino acid sequences - antibodies binding epitope B of αKlothoclone ID name CDR-L3 SEQ ID NO. Generic formula 4804 sb173 QQSSYSLIT 1424807 sb176 QQSSYSLIT 142 4808 sb177 QQSSYSLIT 142 4815 sb184 QQSSYSLIT142 4816 sb185 QQSSYSLIT 142 4821 sb190 QQSSYSLIT 142 4822 sb191QQSSYSLIT 142 4823 sb192 QQSSYSLIT 142 4824 sb193 QQSSYSLIT 142 4825sb194 QQSSYSLIT 142 4827 sb196 QQSSYSLIT 142 4832 sb201 QQSSYSLIT 1424834 sb203 QQSSYSLIT 142 4811 sb180 QQGYALFT 145QQGX₁X₂LX₃T (SEQ ID NO. 126) 4812 sb181 QQGYVVLFT 146wherein X₁ is Y or G; X₂ is W, A or G and X₃ is F or I 4829 sb198QQGGGLIT 153 4813 sb182 QQAAWGGAPIT 147QQAX₁X₂X₃X₄X₅PIT (SEQ ID NO. 127) 4826 sb195 QQAGFFSYPIT 152wherein X₁ is A or G; X₂ is W or F; X₃ is G or F; X₄ is G or S and X₅ is A or Y 4820 sb189 QQSPWGAYLIT 151QQSX₁X₂GX₃YX₄IT (SEQ ID NO. 129) 4833 sb202 QQSSHGHYPIT 156wherein X₁ is S or P; X₂ is H or W; X₃ is H or A and X₄ is P or L 4809sb178 QQSGWAYHPIT 144 QQSX₁X₂X₃YHX₄X₅X₆T (SEQ ID NO. 130) 4805 sb174QQSSGVVYHFLFT 143wherein X₁ is S or G; X₂ is G or W; X₃ is W or A; X₄ is F or P; X₅ is L or I and X₆ is For absent 4818 sb187 QQPYSPIT 149

TABLE 3EHeavy chain CDR-H1 amino acid sequences - antibodies binding epitope B of αKlotho (The carboxyterminal amino acid residue of the sequences listed under column CDR-H1 correspond to IMGT VH domain position 39. Amino acid residues of IMGT CDRs are underlined and amino acid residues of IMGT framework regions are not underlined.Residues at IMGT positions which were randomized in the selection library are bold.)clone ID name CDR-H1 SEQ ID NO. Generic formula A Generic formula B 4813sb182 GFNLYSSS I 166 GFNX₁X₂X₃X₄X₅X₆ (SEQ ID NO. 121) 4825 sb194GFNLYSSS I 166 wherein X₁ is L, F or I; X₂   4811 sb180 GFNLYYSY M 164is Y or S; 4821 sb190 GFNLYYSY M 164 X₃ is S or Y; X₄ is S or Y; X₅ is S4808 sb177 GFNFSSSS I 161 or Y and X₆ is I or M 4815 sb184 GFNFSSSS I161 4816 sb185 GFNFSSSS I 161 4822 sb191 GFNFSSSS I 161 4823 sb192GFNFSSSS I 161 4827 sb196 GFNFSSSS I 161 4832 sb201 GFNFSSSS I 161 4834sb203 GFNFSSSS I 161 4820 sb189 GFNISSYY M 170 4826 sb195 GFNISSYY M 1704809 sb178 GFNIYSYY I 162 GFNIX₁X₂X₃X₄I(SEQ ID NO. 133) 4818 sb187GFNISYSS I 168 wherein X₁ is Y or S; X₂ is Y or S;  4829 sb198 GFNISSYYI 171 X₃ is Y or S and X₄ is Y or S 4804 sb173 GFNLYSYS I 157GFNLX₁X₂X₃X₄X₅(SEQ ID NO. 134) 4805 sb174 GFNLSYYS M 158Wherein X₁ is Y or S; X₂ is Y or S; X₃ 4807 sb176 GFNLSSSY M 160is Y or S; X₄ is Y or S and X₅ is M  4812 sb181 GFNLSSYS M 165 or I 4833sb202 GFNLSSYY M 174 4824 sb193 GFNLSSSS I 163

TABLE 3F Heavy chain CDR-H2 amino acid sequences - antibodies binding epitope B of αKlotho (The aminoterminal residue and the carboxy terminal residue of the sequences listed under column CDR-H2 correspond to IMGT VHdomain positions 55 and 66, respectively. Amino acid residues of IMGT CDRs are underlined and amino acid residues ofIMGT framework regions are not underlined.) clone ID name CDR-H2SEQ ID NO. Generic formula A Generic formula B 4804 sb173 YISSSSGSTY 175YIX₁SSX₂GX₃TX₄(SEQ ID NO. 135) X₁IX₂X₃X₄X₅X₆X₇TX₈ (SEQ ID NO. 122) 4823sb192 YIYSSYGYTY 188 wherein X₁ is S or Y; X₂ is wherein X₁ is Y or S; X₂ is Y or  4827 sb196 YISSSSGSTS 190S or Y; X₃ is S S; X₃ is S or P; X₄ 4832 sb201 YISSSYGYTS 194or Y; X₄ is S or Y is S or Y; X₅ is S or Y; 4808 5b177 SISSSYGYTY 179X₆ is G or S; X₇ is Y or S 4815 5b184 SISSSYGYTY 179 and X₈ is Y or S4816 5b185 SISSSYGYTY 179 4822 sb191 SISSSYGYTY 179 4824 5b193SISSSYGYTY 179 4826 5b195 SISSSYGYTY 179 4834 5b203 SISSSYGYTY 179 48135b182 SISPYSGYTY 184 4818 5b187 SISPYSGYTY 184 4805 5b174 SISSYYGSTY 176SIX₁X₂X₃X₄X₅X₆TX₇(SEQ ID NO. 136) 4807 sb176 SIYPSYGSTS 178wherein X₁ is S or Y; X₂  4809 5b178 SIYSYYGSTS 180  is S or P; X₃ is Y4811 sb180 SIYSSSSYTS 182 or S; X₄ is Y or S; X₅ is 4812 sb181SISSYSGYTS 183 G or S; X₆ is S or Y 4820 5b189 SIYSSYSSTY 186and X₇ = Y or S 4821 sb190 SISPYSGSTY 187 4825 5b194 SISPYYSYTY 189 48295b198 SIYSSYGYTS 191 4833 5b202 SIYPSYSSTY 195

TABLE 3GLiqht chain CDR-L3 amino acid sequences - antibodies bindinq epitope C of αKlotho (Residues at IMGT positions which were randomized in the selection library are bold).clone ID name CDR-L3 SEQ ID NO. Generic formula 4819 sb188 QQGSYYWWSPIT150 QQX₁SX₂YX₃X₄SPIT (SEQ ID NO. 123) 4830 sb199 QQYSVVYWYSPIT 154wherein X₁ is G or Y; X₂ is Y or W; X₃ is W or Y and  X₄ is W, Y or A4831 sb200 QQYSYYYASPIT 155 4814 sb183 QQSSPPIT 148

TABLE 3HHeavy chain CDR-H1 amino acid sequences - antibodies binding epitope C of αKlotho (The carboxyterminal amino acid residue of the sequences listed under column CDR-H1 corresponds to IMGT VH domain position 39. Aminoacid residues of IMGT CDR-H1 are underlined and amino acid residues of IMGT framework regions are notunderlined. Residues at IMGT positions which were randomized in the selection library are bold.)clone ID name CDR-H1 SEQ ID NO. Generic formula 4830 sb199 GFNIYYSS I172 GFNX₁YX₂X₃SX₄(SEQ ID NO. 124) 4831 sb200 GFNIYSSS I 173wherein X₁ is I or L, X₂ is Y or S; X₃ is S or Y and X₄ is I or M 4819sb188 GFNIYSYS M 169 4814 sb183 GFNLYSYS M 167

TABLE 3IHeavy chain CDR-H2 amino acid sequences - antibodies binding epitope C of αKlotho (The amino terminalresidue and the carboxy terminal residue of the sequences listed under column CDR-H2 correspond to IMGT VH domain positions 55 and 66, respectively. Amino acid residues of IMGT CDR-H2 are underlined and amino acid residues of IMGTframework regions are not underlined.) clone ID name CDR-H2 SEQ ID NO.Generic formula 4819 sb188 SIYPSSSYTY 185SIX₁PX₂X₃X₄YTY (SEQ ID NO. 125) 4830 sb199 SIYPYYSYTY 192wherein X₁ is S or Y, X₂ is S or Y; X₃ is S or Y and X₄ is S or G 4831sb200 SIYPYSGYTY 193 4814 sb183 SISPYSGYTY 184

TABLE 4Example of full length sequences for additional αKlotho antibodiesLight chain (hK) amino acid sequence: SEQ ID NO: 317DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAVVYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSYSLITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLight Chain (hK) nucleic acid sequence: SEQ ID NO: 318GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGTCCGTGTCCAGCGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACTCGGCATCCAGCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAATCTTCTTATTCTCTGATCACGTTCGGACAGGGTACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTHeavy chain (hG1) amino acid sequence: SEQ ID NO: 319EVQLVESGGGLVQPGGSLRLSCAASGFNLYSYS IHVVVRQAPGKGLEVVVAY ISSSSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGWGGGYWFYPVYGIDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHeavy chain (hG1) nucleic acid sequence: SEQ ID NO: 320GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCA ACCTCTATTCTTATTCTATCCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCA TATATTTCTTCTTCTTCTGGCTCTACTTATTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCGGTTGGGGTGGTGGTTACTGGTTCTACCCGGTTTACGGTATTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA Underlined identifies CDR sequence; Bold identifiesresidues at IMGT positions which were randomized in the selectionlibrary (Antibody #4804 shown as example here); Italics representconstant domain sequence

TABLE 5 Summary of binding characteristics of additional αKlothoantibodies ID name affinity epitope group 48 sb106 1.9 nM A 4804 sb173770 pM B 4805 sb174 240 pM B 4806 sb175 2.7 nM nd 4807 sb176 6.1 nM B4808 sb177 1.6 nM B 4809 sb178 845 pM B 4810 sb179 1.9 nM nd 4811 sb1802.3 nM B 4812 sb181 885 pM B 4813 sb182 5.2 nM B 4814 sb183 4.7 nM C4815 sb184 360 pM B 4816 sb185 3.7 nM B 4817 sb186 4.1 nM nd 4818 sb1872.1 nM B 4819 sb188 550 pM C 4820 sb189 2.7 nM B 4821 sb190 5.1 nM B4822 sb191 8.7 nM B 4823 sb192 1.6 nM B 4824 sb193 3.5 nM B 4825 sb194nd B 4826 sb195 3.3 nM B 4827 sb196 5.6 nM B 4828 sb197 4.9 nM nd 4829sb198 1.8 nM B 4830 sb199 2.9 nM C 4831 sb200 1.6 nM C 4832 sb201 515 pMB 4833 sb202 1.4 nM B 4834 sb203 1.1 nM B

While the present application has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the application is not limited to the disclosedexamples. To the contrary, the application is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. Specifically, the sequences associated with eachaccession numbers provided herein including for example accessionnumbers and/or biomarker sequences (e.g. protein and/or nucleic acid)provided in the Tables or elsewhere, are incorporated by reference inits entirely.

CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION

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1. An antibody and/or binding fragment thereof, wherein the antibody orbinding fragment thereof comprises a light chain variable region and aheavy chain variable region, the light chain variable region comprisingcomplementarity determining region (CDR) CDR-L3 and the heavy chainvariable region comprising CDR-H1, CDR-H2 and CDR-H3, with the aminoacid sequences of said CDRs comprising one or more of the sequences setforth below: CDR-L3; selected from any one of SEQ ID NOs: 123, 126-130,142, 148 or 149; CDR-H1: SEQ ID NOs: 121 or 124; CDR-H2; SEQ ID NOs: 122or 125; and/or CDR-H3: selected from any one of SEQ ID NOs: 196-226. 2.The antibody and/or binding fragment thereof of claim 1, wherein theCDRs comprise the amino acid sequences selected from SEQ ID NOs:142-226, optionally as set forth below: Light chain variable region:CDR-L3: selected from any one of SEQ ID NOs: 142-156; Heavy chainvariable region: CDR-H1: selected from any one of SEQ ID NOs: 157-174;CDR-H2: selected from any one of SEQ ID NOs: 175-195; and/or CDR-H3:selected from any one of SEQ ID NOs: 196-226.
 3. The antibody and/orbinding fragment thereof of claim 1 or 2, wherein the light chainvariable region comprises CDR-L1 and/or CDR-L2 comprising the amino acidsequences set forth below: CDR-L1: SEQ ID NO: 140; and/or CDR-L2: SEQ IDNO:
 141. 4. The antibody and/or binding fragment thereof of any ofclaims 1 to 3, wherein the antibody and/or binding fragment thereof isselected from the group consisting of a monoclonal antibody, animmunoglobulin molecule, a Fab, a Fab′, a F(ab)2, a F(ab′)2, a Fv, adisulfide linked Fv, a scFv, a disulfide linked scFv, a single chaindomain antibody, scFab, a diabody, a dimer, a minibody, a bispecificantibody fragment, a chimeric antibody, a humanized antibody and apolyclonal antibody.
 5. The antibody and/or binding fragment thereof ofany one of claims 1 to 4, wherein the αKlotho polypeptide is mammalianαKlotho polypeptide.
 6. The antibody and/or binding fragment thereof ofclaim 5, wherein the mammalian αKlotho polypeptide is human αKlothopolypeptide.
 7. The antibody and/or binding fragment thereof of claim 5,wherein the mammalian αKlotho is rodent αKlotho polypeptide.
 8. Theantibody and/or binding fragment thereof of claim 7, wherein the rodentαKlotho polypeptide is mouse αKlotho polypeptide or rat αKlothopolypeptide.
 9. The antibody and/or binding fragment thereof of any oneof claims 1 to 8, wherein the αKlotho polypeptide is folded αKlothopolypeptide and/or soluble αKlotho polypeptide.
 10. The antibody and/orbinding fragment thereof of claim 9, wherein the antibody and/or bindingfragment thereof binds soluble folded αKlotho polypeptide found inurine, plasma, and/or serum.
 11. The antibody and/or binding fragmentthereof of any one of claims 1 to 10, wherein the antibody and/orbinding fragment thereof binds a complex comprising folded αKlothopolypeptide.
 12. The antibody and/or binding fragment thereof accordingto claim 11, wherein the folded αKlotho polypeptide forms a complex witha fibroblast growth factor (FGF) receptor, optionally FGFRI c.
 13. Theantibody and/or binding fragment of any one of claims 1 to 12, whereinthe antibody and/or binding fragment is labeled with a detectable tag.14. The antibody and/or binding fragment of claim 13, wherein thedetectable tag is a His-tag, a HA-tag, a GST-tag, or a FLAG-tag.
 15. Anantibody complex comprising the antibody and/or binding fragment thereofof any one of claims 1 to 14 and αKlotho polypeptide, optionally furthercomprising FGFRI c.
 16. The antibody complex of claim 15 furthercomprising an antibody that binds epitope A.
 17. A nucleic acid encodingan antibody and/or binding fragment thereof comprising a light chainvariable region and a heavy chain variable region, the light chainvariable region comprising CDR-L3 and the heavy chain variable regioncomprising CDR-H1, CDR-H2 and CDR-H3, with the amino acid sequences ofsaid CDRs comprising one or more of the sequences set forth below:CDR-L3; selected from any one of SEQ ID NOs: 123, 126-130, 142, 148 or149; CDR-H1: SEQ ID NOs: 121 or 124; CDR-H2; SEQ ID NOs: 122 or 125;and/or CDR-H3: selected from any one of SEQ ID NOs: 196-226.
 18. Thenucleic acid of claim 17, comprising nucleic acid sequences selectedfrom SEQ ID NOs: 229-316, wherein: CDR-L3: selected from any one of SEQID NOs: 229-243; CDR-H1 selected from any one of SEQ ID NOs: 244-262;CDR-H2 selected from any one of SEQ ID NOs: 263-285; and/or CDR-H3selected from any one of SEQ ID NOs: 286-316.
 19. The nucleic acid ofclaim 17 or 18, wherein the light chain variable region comprises CDR-and/or CDR-L2 comprising the nucleic acid sequences set forth below:CDR-L1: SEQ ID NO: 227 and/or CDR-L2: SEQ ID NO:
 228. 20. A vectorcomprising the nucleic acid of any one of claims 17 to
 19. 21. Arecombinant cell producing the antibody and/or binding fragment thereofof any one of claims 1 to 16, comprising the nucleic acid of any one ofclaims 17 to 19 or the vector of claim
 20. 22. A composition comprisingthe antibody and/or binding fragment thereof of any one of claims 1 to16, the nucleic acid of any one of claims 17 to 19, the vector of claim20 or the recombinant cell of claim
 21. 23. A method for producing anantibody and/or binding fragment thereof, the steps comprising: a.expressing in a host cell a nucleic acid encoding amino acid sequencesof the antibody and/or binding fragment thereof according to any one ofclaims 1 to 16; b. culturing the host cell to produce the antibodyand/or binding fragment thereof; and c. isolating and purifying theantibody and/or binding fragment thereof from the host cell.
 24. Themethod of claim 23, wherein the nucleic acid comprises nucleic acidsequences selected from SEQ ID NOs: 229-316, wherein: CDR-L3: SEQ IDNOs: 229-243; CDR-H1 SEQ ID NOs: 244-262; CDR-H2 SEQ ID NOs: 263-285;and/or CDR-H3 SEQ ID NOs: 286-316.
 25. An immunoassay comprising theantibody and/or binding fragment thereof of any one of claims 1 to 16.26. The immunoassay of claim 25 wherein the immunoassay is an enzymelinked immunosorbent assay (ELISA).
 27. The immunoassay of claim 26,wherein the ELISA is a sandwich ELISA comprising a capture antibody anda detection antibody, wherein the capture antibody and/or detectionantibody is an antibody and/or binding fragment thereof of any one ofclaims 1 to
 16. 28. The immunoassay of claim 27, wherein the captureantibody and the detection antibody are selected from an antibody and/orbinding fragment thereof of any one of claims 1 to 16, and an antibodyhaving light and heavy chain variable regions comprising the amino acidsequences of SEQ ID NO: 11 and 12, respectively.
 29. The immunoassay ofclaim 27 or 28, wherein one of the capture and detection antibodies isan antibody and/or binding fragment thereof any one of claims 1 to 16and the other of the capture and detection antibodies is an antibodyhaving light and heavy chain variable regions comprising the amino acidsequences of SEQ ID NO: 11 and 12, respectively.
 30. The immunoassay ofany one of claims 27 to 29 for the detection and/or measuring of αKlothopolypeptide in a sample, wherein the method of making the immunoassaycomprises: a. coating a solid support with the capture antibody; b.contacting the capture antibody with the sample under conditions to forma capture antibody:αKlotho complex; c. removing unbound sample; d.contacting the capture antibody:αKlotho complex with the detectionantibody; e. removing unbound detection antibody; and f. detectingand/or measuring the capture antibody:αKlotho complex.
 31. Theimmunoassay of claim 26, wherein the ELISA is a competitive ELISA. 32.An assay for detecting and/or measuring level of αKlotho polypeptide ina sample, the assay comprising: a. contacting a sample with the antibodyand/or binding fragment thereof of any one of claims 1 to 16 underconditions to form an antibody:αKlotho complex; and b. detecting and/ormeasuring the antibody:αKlotho complex.
 33. An assay for detectingand/or measuring soluble αKlotho the method comprising: a. contacting asample, the sample being a biological fluid, with the antibody and/orbinding fragment thereof of any one of claims 1 to 16 under conditionsto form an antibody: soluble αKlotho complex; and b. detecting and/ormeasuring the antibody: soluble αKlotho complex.
 34. The assay of claim32 or 33, wherein the antibody:αKlotho complex is detected byimmunoprecipitation, immunoblot, immunohistochemistry,immunocytochemistry and fluorescence-activated cell sorting (FACS). 35.A method for screening, for diagnosing or for detecting a kidneycondition selected from chronic kidney disease (CKD) and acute kidneyinjury (AKI) in a subject, the method comprising: a. measuring the levelof αKlotho in a sample from a subject optionally using an antibody orfragment thereof of any one of claims 1 to 16 or using the assay of anyone of claims 25 to 34; and b. comparing the level of αKlotho in thesample with a control, wherein a decreased level of αKlotho in thesample compared to the control is indicative that the subject has akidney condition selected from CKD or AKI.
 36. The method of claim 35,wherein the CKD is early CKD, optionally stage 1 or stage 2, or stage 3,stage 4, stage 5 or stage
 6. 37. The method of claim 35 or 36 whereinthe sample is selected from a fresh tissue sample, a frozen sample and afixed sample such as a mildly fixed sample.
 38. The method of any one ofclaims 35 to 37, wherein the level of αKlotho is determined byimmunoprecipitation.
 39. A method for prognosing a recovery after AKI,the method comprising: a. determining the level of αKlotho in a samplefrom a subject optionally using the antibody or fragment thereof of anyone of claims 1 to 16 or using the assay of any one of claims 25 to 34;and b. comparing the level of αKlotho in the sample with a control,wherein an increased level of αKlotho in the sample compared to thecontrol is indicative that the subject has a higher likelihood ofrecovery after AKI.
 40. A method for prognosing long term complicationsafter AKI, the method comprising: a. determining the level of αKlotho ina sample from a subject optionally using the antibody or fragmentthereof of any one of claims 1 to 16 or using the assay of any one ofclaims 25 to 34; and b. comparing the level of αKlotho in the samplewith a control, wherein an increased level of αKlotho in the samplecompared to the control is indicative that the subject has a higherlikelihood of fewer long term complications after AKI.
 41. A method forprognosing the rate of progression of CKD, the method comprising: a.determining the level of αKlotho in a sample from a subject optionallyusing the antibody or fragment thereof of any one of claims 1 to 16 orusing the assay of any one of claims 25 to 34; and b. comparing thelevel of αKlotho in the sample with a control, wherein an increasedlevel of αKlotho in the sample compared to the control is indicativethat the subject has a higher likelihood of a slower rate of progressionof CKD.
 42. A method for prognosing extra-renal complications in CKD,the method comprising: a. determining the level of αKlotho in a samplefrom a subject optionally using the antibody or fragment thereof of anyone of claims 1 to 16 or using the assay of any one of claims 25 to 34;and b. comparing the level of αKlotho in the sample with a control,wherein an increased level of αKlotho in the sample compared to thecontrol is indicative that the subject has a higher likelihood of havingfewer extra-renal complications related to CKD.
 43. A kit comprising theantibody and/or binding fragment thereof according to any one of claims1 to 16, a reference agent and optionally instructions for use thereof.44. The kit of claim 43, further comprising an antibody and/or bindingfragment having light and heavy chain variable regions comprising theamino acid sequences of SEQ ID NO: 11 and 12, respectively.